Methods, compositions, and kits for detecting allelic variants

ABSTRACT

In some embodiments, the present inventions relates generally to compositions, methods and kits for use in discriminating sequence variation between different alleles. More specifically, in some embodiments, the present invention provides for compositions, methods and kits for quantitating rare (e.g., mutant) allelic variants, such as SNPs, or nucleotide (NT) insertions or deletions, in samples comprising abundant (e.g., wild type) allelic variants with high specificity and selectivity. In particular, in some embodiments, the invention relates to a highly selective method for mutation detection referred to as competitive allele-specific TaqMan PCR (“cast-PCR”).

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.12/748,329 filed Mar. 26, 2010, which is continuation-in-part of U.S.application Ser. No. 12/641,321 filed Dec. 17, 2009 and claims thebenefit of priority under 35 U.S.C. 119 to U.S. Provisional ApplicationNos. 61/138,521 filed Dec. 17, 2008; 61/258,582 filed Nov. 5, 2009;61/253,501 filed Oct. 20, 2009; 61/251,623 filed Oct. 14, 2009;61/186,775 filed Jun. 12, 2009; and 61/164,230 filed Mar. 27, 2009, allof which are incorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety.

BACKGROUND

Single nucleotide polymorphisms (SNPs) are the most common type ofgenetic diversity in the human genome, occurring at a frequency of aboutone SNP in 1,000 nucleotides or less in human genomic DNA (Kwok, P-Y,Ann Rev Genom Hum Genet 2001, 2: 235-258). SNPs have been implicated ingenetic disorders, susceptibility to different diseases, predispositionto adverse reactions to drugs, and for use in forensic investigations.Thus, SNP (or rare mutation) detection provides great potentials indiagnosing early phase diseases, such as detecting circulating tumorcells in blood, for prenatal diagnostics, as well as for detection ofdisease-associated mutations in a mixed cell population.

Numerous approaches for SNP genotyping have been developed based onmethods involving hybridization, ligation, or DNA polymerases (Chen, X.,and Sullivan, P F, The Pharmacogeonomics Journal 2003, 3, 77-96.). Forexample, allele-specific polymerase chain reaction (AS-PCR) is a widelyused strategy for detecting DNA sequence variation (Wu D Y, Ugozzoli L,Pal B K, Wallace R B., Proc Natl Acad Sci USA 1989; 86:2757-2760).AS-PCR, as its name implies, is a PCR-based method whereby one or bothprimers are designed to anneal at sites of sequence variations whichallows for the ability to differentiate among different alleles of thesame gene. AS-PCR exploits the fidelity of DNA polymerases, which extendprimers with a mismatched 3′ base at much lower efficiency, from 100 to100,000 fold less efficient, than that with a matched 3′ base (Chen, X.,and Sullivan, P F, The Pharmacogeonomics Journal 2003; 3:77-96). Thedifficulty in extending mismatched primers results in diminished PCRamplification that can be readily detected.

The specificity and selectivity of AS-PCR, however, is largely dependenton the nature of exponential amplification of PCR which makes the decayof allele discriminating power rapid. Even though primers are designedto match a specific variant to selectively amplify only that variant, inactuality significant mismatched amplification often occurs. Moreover,the ability of AS-PCR to differentiate between allelic variants can beinfluenced by the type of mutation or the sequence surrounding themutation or SNP (Ayyadevara S, Thaden J J, Shmookler Reis R J., AnalBiochem 2000; 284:11-18), the amount of allelic variants present in thesample, as well as the ratio between alternative alleles. Collectively,these factors are often responsible for the frequent appearance offalse-positive results, leading many researchers to attempt to increasethe reliability of AS-PCR (Orou A, Fechner B, Utermann G, Menzel H J.,Hum Mutat 1995; 6:163-169) (Imyanitov E N, Busboy K G, Suspitsin E N,Kuligina E S, Belogubova E V, Grigoriev M Y, et al., Biotechniques 2002;33:484-490) (McKinzie P B, Parsons B L. Detection of rare K-ras codon 12mutations using allele-specific competitive blocker PCR. Mutat Res 2002;517:209-220) (Latorra D, Campbell K, Wolter A, Hurley J M., Hum Mutat2003; 22:79-85).

In some cases, the selectivity of AS-PCR has been increased anywherefrom detection of 1 in 10 alleles to 1 in 100,000 alleles by usingSNP-based PCR primers containing locked nucleic acids (LNAs) (Latorra,D., et al., Hum Mut 2003, 2:79-85; Nakiandwe, J. et al., Plant Method2007, 3:2) or modified bases (Koizumi, M. et al. Anal Biochem. 2005,340:287-294). However, these base “mimics” or modifications increase theoverall cost of analysis and often require extensive optimization.

Another technology involving probe hybridization methods used fordiscriminating allelic variations is TaqMan® genotyping. However, likeAS-PCR, selectivity using this method is limited and not suitable fordetecting rare (1 in ≥1,000) alleles or mutations in a mixed sample.

SUMMARY

In some embodiments, the present inventions relates generally tocompositions, methods and kits for use in discriminating sequencevariation between different alleles. More specifically, in someembodiments, the present invention provides for compositions, methodsand kits for quantitating rare (e.g., mutant) allelic variants, such asSNPs, or nucleotide (NT) insertions or deletions, in samples comprisingabundant (e.g., wild type) allelic variants with high specificity. Inparticular, in some embodiments, the invention relates to a highlyselective method for mutation detection referred to as competitiveallele-specific TaqMan PCR (“cast-PCR”).

In one aspect, the present invention provides compositions for use inidentifying and/or quantitating allelic variants in nucleic acidsamples. Some of these compositions can comprise: (a) an allele-specificprimer; (b) an allele-specific blocker probe; (c) a detector probe;and/or (d) a locus-specific primer.

In some embodiments of the compositions, the allele-specific primercomprises a target-specific portion and an allele-specific nucleotideportion. In some embodiments, the allele-specific primer may furthercomprise a tail. In some exemplary embodiments, the tail is located atthe 5′ end of the allele-specific primer. In other embodiments, the tailof the allele-specific primer has repeated guanine and cytosine residues(“GC-rich”). In some embodiments, the melting temperature (“Tm”) of theentire allele-specific primer ranges from about 50° C. to 67° C. In someembodiments, the allele-specific primer concentration is between about20-900 nM.

In some embodiments of the compositions, the allele-specific nucleotideportion of the allele-specific primer is located at the 3′ terminus. Insome embodiments, the selection of the allele-specific nucleotideportion of the allele-specific primer involves the use of a highlydiscriminating base (e.g., for detection of A/A, A/G, G/A, G/G, A/C, orC/A alleles). In some embodiments, for example when the allele to bedetected involves A/G or C/T SNPs, A or G is used as the 3′allele-specific nucleotide portion of the allele-specific primer (e.g.,if A or T is the target allele), or C or T is used as the 3′allele-specific nucleotide portion of the allele-specific primer (e.g.,if C or G is the target allele). In other embodiments, A is used as thediscriminating base at the 3′ end of the allele-specific primer whendetecting and/or quantifying A/T SNPs. In other embodiments, G is usedas the discriminating base at the 3′ end of the allele-specific primerwhen detecting and/or quantifying C/G SNPs.

In some embodiments of the compositions, the allele-specific blockerprobe comprises a non-extendable blocker moiety at the 3′ terminus. Insome exemplary embodiments, the non-extendable blocker moiety is a minorgroove binder (MGB). In some embodiments, the target allele position islocated about 6-10, such as about 6, about 7, about 8, about 9, or about10 nucleotides away from the non-extendable blocker moiety of theallele-specific blocker probe. In some embodiments, the allele-specificblocker probe comprises an MGB moiety at the 5′ terminus. In someexemplary embodiments, the allele-specific blocker probe is not cleavedduring PCR amplification. In some embodiments, the Tm of theallele-specific blocker probe ranges from about 58° C. to 66° C.

In some embodiments of the compositions, the allele-specific blockerprobe and/or allele-specific primer comprise at least one modified base.In some embodiments, the modified base(s) may increase the difference inthe Tm between matched and mismatched target sequences and/or decreasemismatch priming efficiency, thereby improving not only assayspecificity bust also selectivity. Such modified base(s) may include,for example, 8-Aza-7-deaza-dA (ppA), 8-Aza-7-deaza-dG (ppG),2′-Deoxypseudoisocytidine (iso dC), 5-fluoro-2′-deoxyuridine (fdU),locked nucleic acid (LNA), or 2′-O,4′-C-ethylene bridged nucleic acid(ENA) bases (also see, for example, FIG. 4B). In some embodiments themodified base is located at (a) the 3′-end, (b) the 5′-end, (c) at aninternal position or at any combination of (a), (b) or (c) within theallele-specific blocker probe and/or the allele-specific primer.

In some embodiments of the compositions, the detector probe is asequence-based or locus-specific detector probe. In other embodimentsthe detector probe is a 5′ nuclease probe. In some exemplaryembodiments, the detector probe can comprises an MGB moiety, a reportermoiety (e.g., FAM™, TET™, JOE™, VIC™, or SYBR® Green), a quencher moiety(e.g., Black Hole Quencher™ or TAMRA™), and/or a passive reference(e.g., ROX™). In some exemplary embodiments, the detector probe isdesigned according to the methods and principles described in U.S. Pat.No. 6,727,356 (the disclosure of which is incorporated herein byreference in its entirety). In some exemplary embodiments, the detectorprobe is a TaqMan® probe (Applied Biosystems, Foster City).

In some embodiments of the compositions, the composition can furthercomprise a polymerase; deoxyribonucleotide triphosphates (dNTPs); otherreagents and/or buffers suitable for amplification; and/or a templatesequence or nucleic acid sample. In some embodiments, the polymerase canbe a DNA polymerase. In some other embodiments, the polymerase can bethermostable, such as Taq DNA polymerase. In other embodiments, thetemplate sequence or nucleic acid sample can be DNA, such as genomic DNA(gDNA) or complementary DNA (cDNA). In other embodiments the templatesequence or nucleic acid sample can be RNA, such as messenger RNA(mRNA).

In another aspect, the present disclosure provides methods foramplifying an allele-specific sequence. Some of these methods caninclude one or more of the following: (a) hybridizing an allele-specificprimer to a first nucleic acid molecule comprising a first allele(allele-1); (b) hybridizing an allele-specific blocker probe to a secondnucleic acid molecule comprising a second allele (allele-2), whereinallele-2 corresponds to the same loci as allele-1; (c) hybridizing adetector probe to the first nucleic acid molecule; (d) hybridizing alocus-specific primer to the extension product of the allele-specificprimer; and (e) PCR amplifying the first nucleic acid moleculecomprising allele-1.

In another aspect, the present invention provides methods for detectingand/or quantitating an allelic variant in a pooled or mixed samplecomprising other alleles. Some of these methods can include one or moreof the following: (a) in a first reaction mixture hybridizing a firstallele-specific primer to a first nucleic acid molecule comprising afirst allele (allele-1) and in a second reaction mixture hybridizing asecond allele-specific primer to a first nucleic acid moleculecomprising a second allele (allele-2), wherein allele-2 corresponds tothe same locus as allele-1; (b) in the first reaction mixturehybridizing a first allele-specific blocker probe to a second nucleicacid molecule comprising allele-2 and in the second reaction mixturehybridizing a second allele-specific blocker probe to a second nucleicacid molecule comprising allele-1; (c) in the first reaction mixture,hybridizing a first detector probe to the first nucleic acid moleculeand in the second reaction mixture and hybridizing a second detectorprobe to the first nucleic acid molecule; (d) in the first reactionmixture hybridizing a first locus-specific primer to the extensionproduct of the first allele-specific primer and in the second reactionmixture hybridizing a second locus-specific primer to the extensionproduct of the second allele-specific primer; and (e) PCR amplifying thefirst nucleic acid molecule to form a first set or sample of ampliconsand PCR amplifying the second nucleic acid molecule to form a second setor sample of amplicons; and (f) comparing the first set of amplicons tothe second set of amplicons to quantitate allele-1 in the samplecomprising allele-2 and/or allele-2 in the sample comprising allele-1.

In some embodiments of the methods, the first and/or secondallele-specific primer comprises a target-specific portion and anallele-specific nucleotide portion. In some embodiments, the firstand/or second allele-specific primer may further comprise a tail. Insome embodiments, the Tm of the entire first and/or secondallele-specific primer ranges from about 50° C. to 67° C. In someembodiments the first and/or second allele-specific primer concentrationis between about 20-900 nM.

In some embodiments of the methods, the target-specific portion of thefirst allele-specific primer and the target-specific portion of thesecond allele-specific primer comprise the same sequence. In otherembodiments, the target-specific portion of the first allele-specificprimer and the target-specific portion of the second allele-specificprimer are the same sequence.

In some embodiments of the methods, the tail is located at the 5′-end ofthe first and/or second allele-specific primer. In some embodiments, the5′ tail of the first allele-specific primer and the 5′ tail of thesecond allele-specific primer comprise the same sequence. In otherembodiments, the 5′ tail of the first allele-specific primer and the 5′tail of the second allele-specific primer are the same sequence. Inother embodiments, the tail of the first and/or second allele-specificprimer is GC-rich.

In some embodiments of the methods, the allele-specific nucleotideportion of the first allele-specific primer is specific to a firstallele (allele-1) of a SNP and the allele-specific nucleotide portion ofthe second allele-specific primer is specific to a second allele(allele-2) of the same SNP. In some embodiments of the methods, theallele-specific nucleotide portion of the first and/or secondallele-specific primer is located at the 3′-terminus. In someembodiments, the selection of the allele-specific nucleotide portion ofthe first and/or second allele-specific primer involves the use of ahighly discriminating base (e.g., for detection of A/A, A/G, G/A, G/G,A/C, or C/A alleles). In some embodiments, for example when the alleleto be detected involves A/G or C/T SNPs, A or G is used as the 3′allele-specific nucleotide portion of the first and/or secondallele-specific primer (e.g., if A or T is the major allele), or C or Tis used as the 3′ allele-specific nucleotide portion of the first and/orsecond allele-specific primer (e.g., if C or G is the major allele). Inother embodiments, A is used as the discriminating base at the 3′ end ofthe first and/or second allele-specific primer when detecting and/orquantifying A/T SNPs. In other embodiments, G is used as thediscriminating base at the 3′ end of the first and/or secondallele-specific primer when detecting and/or quantifying C/G SNPs.

In some embodiments of the methods, the first and/or secondallele-specific blocker probe comprises a non-extendable blocker moietyat the 3′ terminus. In some exemplary embodiments, the non-extendableblocker moiety is an MGB. In some embodiments, the target alleleposition is located about 6-10, such as about 6, about 7, about 8, about9, or about 10 nucleotides away from the non-extendable blocker moietyof the first and/or second allele-specific blocker probe. In someembodiments, the first and/or second allele-specific blocker probecomprises an MGB moiety at the 5′-terminus. In other embodiments, thefirst and/or second allele-specific blocker probe is not cleaved duringPCR amplification. In some embodiments, the Tm of the first and/orsecond allele-specific blocker probe ranges from about 58° C. to 66° C.

In some embodiments of the methods, the first and/or secondallele-specific blocker probe and/or the first and/or secondallele-specific primer comprises at least one modified base. In someembodiments, the modified base(s) may increase the difference in the Tmbetween matched and mismatched target sequences and/or decrease mismatchpriming efficiency, thereby improving not only assay specificity, butalso selectivity. Such modified base(s) may include, for example,8-Aza-7-deaza-dA (ppA), 8-Aza-7-deaza-dG (ppG),2′-Deoxypseudoisocytidine (iso dC), 5-fluoro-2′-deoxyuridine (fdU),locked nucleic acid (LNA), or 2′-O,4′-C-ethylene bridged nucleic acid(ENA) bases (see also, for example, FIG. 4B). In some embodiments themodified base is located at (a) the 3′-end, (b) the 5′-end, (c) at aninternal position or at any combination of (a), (b) or (c) within saidfirst and/or second allele-specific blocker probe and/or the firstand/or second allele-specific primer.

In some embodiments of the methods, the specificity of allelicdiscrimination is improved by the inclusion of a modified base in thefirst and/or second allele-specific primer and/or first, and/or secondallele-specific blocker probe as compared to the use of a non-modifiedallelic-specific primer or blocker probe. In some embodiments, theimprovement in specificity is at least 2 fold.

In some embodiments of the methods, the specificity of allelicdiscrimination is at least 2 fold better than the specificity of allelicdiscrimination using Allele-Specific PCR with a Blocking reagent(ASB-PCR) methods.

In some embodiments, the methods further comprise a 2-stage cyclingprotocol. In some embodiments, the number of cycles in the first stageof the 2-stage cycling protocol comprises fewer cycles than the numberof cycles used in the second stage. In other embodiments, the number ofcycles in the first stage is about 90% fewer cycles than the number ofcycles in the second stage. In yet other embodiments, the number ofcycles in the first stage is between 3-7 cycles and the number of cyclesin the second stage is between 42-48 cycles.

In some embodiments, the annealing/extension temperature used during thefirst cycling stage of the 2-stage cycling protocol is between 1-3° C.lower than the annealing/extension temperature used during the secondstage. In preferred embodiments, the annealing/extension temperatureused during the first cycling stage of the 2-stage cycling protocol isbetween 56-59° C. and the annealing/extension temperature used duringthe second stage is between 60-62° C.

In some embodiments, the methods further comprise a pre-amplificationstep. In preferred embodiments, the pre-amplification step comprises amultiplex amplification reaction that uses at least two complete sets ofallele-specific primers and locus-specific primers, wherein each set issuitable or operative for amplifying a specific polynucleotide ofinterest. In other embodiments, the products of the multiplexamplification reaction are divided into secondary single-plexamplification reactions, such as a cast-PCR reaction, wherein eachsingle-plex reaction contains at least one primer set previously used inthe multiplex reaction. In other embodiments, the multiplexamplification reaction further comprises a plurality of allele-specificblocker probes. In some embodiments, the multiplex amplificationreaction is carried out for a number of cycles suitable to keep thereaction within the linear phase of amplification.

In some embodiments of the methods, the first and/or second detectorprobes are the same. In some embodiments, the first and/or seconddetector probes are different. In some embodiments, the first and/orsecond detector probe is a sequence-based or locus-specific detectorprobe. In other embodiments the first and/or second detector probe is a5′ nuclease probe. In some exemplary embodiments, the first and/orsecond detector probes comprises an MGB moiety, a reporter moiety (e.g.,FAM™, TET™, JOE™, VIC™, or SYBR® Green), a quencher moiety (e.g., BlackHole Quencher™ or TAMRA™), and/or a passive reference (e.g., ROX™). Insome exemplary embodiments, the first and/or second detector probe isdesigned according to the methods and principles described in U.S. Pat.No. 6,727,356 (the disclosure of which is incorporated herein byreference in its entirety). In some exemplary embodiments, the detectorprobe is a TaqMan® probe.

In some embodiments of the methods, the first locus-specific primer andthe second locus-specific primer comprise the same sequence. In someembodiments the first locus-specific primer and the secondlocus-specific primer are the same sequence.

In some embodiments of the methods, the first and/or second reactionmixtures can further comprises a polymerase; dNTPs; other reagentsand/or buffers suitable for PCR amplification; and/or a templatesequence or nucleic acid sample. In some embodiments, the polymerase canbe a DNA polymerase. In some embodiments, the polymerase can bethermostable, such as Taq DNA polymerase. In some embodiments, thetemplate sequence or nucleic acid sample can be DNA, such as gDNA orcDNA. In other embodiments the template sequence or nucleic acid samplecan be RNA, such as mRNA.

In some embodiments of the methods, the first allele-specific blockerprobe binds to the same strand or sequence as the second allele-specificprimer, while the second allele-specific blocker probe binds to the samestrand or sequence as the first allele-specific primer. In someembodiments, the first and/or second allele-specific blocker probes areused to reduce the amount of background signal generated from either thesecond allele and/or the first allele, respectively. In someembodiments, first and/or second allele-specific blocker probes arenon-extendable and preferentially anneal to either the second allele orthe first allele, respectively, thereby blocking the annealing of, forexample, the extendable first allele-specific primer to the secondallele and/or the extendable second allele-specific primer to firstallele.

In some exemplary embodiments, the first allele is a rare (e.g., minor)or mutant allele. In other exemplary embodiments the second allele is anabundant (e.g., major) or wild type allele.

In another aspect, the present invention provides kits for quantitatinga first allelic variant in a sample comprising a second allelic variantinvolving: (a) a first allele-specific primer; (b) a secondallele-specific primer; (c), a first locus-specific primer; (d) a secondlocus-specific primer; (e) a first allele-specific blocker probe; (f) asecond allele-specific blocker probe; and (g) a first locus-specificdetector probe and (h) a second locus-specific detector probe.

In some embodiments of the kits, the first and/or second allele-specificprimer comprises a target-specific portion and an allele-specificnucleotide portion. In some embodiments, the first and/or secondallele-specific primer may further comprise a tail. In some embodiments,the Tm of the entire first and/or second allele-specific primer rangesfrom about 50° C. to 67° C. In some embodiments the first and/or secondallele-specific primer concentrations are between about 20-900 nM.

In some embodiments of the kits, the target-specific portion of thefirst allele-specific primer and the target-specific portion of thesecond allele-specific primer comprise the same sequence. In otherembodiments, the target-specific portion of the first allele-specificprimer and the target-specific portion of the second allele-specificprimer are the same sequence.

In some embodiments of the kits, the tail is located at the 5′ end ofthe first and/or second allele-specific primer. In some embodiments, the5′ tail of the first allele-specific primer and the 5′ tail of thesecond allele-specific primer comprise the same sequence. In otherembodiments, the 5′ tail of the first allele-specific primer and the 5′tail of the second allele-specific primer are the same sequence. Inother embodiments, the tail of the first and/or second allele-specificprimer is GC rich.

In some embodiments of the kits, the allele-specific nucleotide portionof the first allele-specific primer is specific to a first allele(allele-1) of a SNP and the allele-specific nucleotide portion of thesecond allele-specific primer is specific to a second allele (allele-2)of the same SNP. In some embodiments of the disclosed methods, theallele-specific nucleotide portion of the first and/or secondallele-specific primer is located at the 3′ terminus. In someembodiments, the selection of the allele-specific nucleotide portion ofthe first and/or second allele-specific primer involves the use of ahighly discriminating base (e.g., for detection of A/A, A/G, G/A, G/G,A/C, or C/A alleles) (FIG. 2). In some embodiments, for example when theallele to be detected involves A/G or C/T SNPs, A or G is used as the 3′allele-specific nucleotide portion of the first and/or secondallele-specific primer (e.g., if A or T is the major allele), or C or Tis used as the 3′ allele-specific nucleotide portion of the first and/orsecond allele-specific primer (e.g., if C or G is the major allele). Inother embodiments, A is used as the discriminating base at the 3′ end ofthe first and/or second allele-specific primer when detecting and/orquantifying A/T SNPs. In other embodiments, G is used as thediscriminating base at the 3′ end of the first and/or secondallele-specific primer when detecting and/or quantifying C/G SNPs.

In some embodiments of the kits, the first and/or second allele-specificblocker probe comprises a non-extendable blocker moiety at the 3′terminus. In some exemplary embodiments, the non-extendable blockermoiety is an MGB. In some embodiments, the target allele position islocated about 6-10, such as about 6, about 7, about 8, about 9, or about10 nucleotides away from the non-extendable blocker moiety of the firstand/or second allele-specific blocker probe. In some embodiments, thefirst and/or second allele-specific blocker probe comprises an MGBmoiety at the 5′ terminus. In other embodiments, the first and/or secondallele-specific blocker probe is not cleaved during PCR amplification.In some embodiments, the Tm of the first and/or second allele-specificblocker probe ranges from about 58° C. to 66° C.

In some embodiments of the kits, the allele-specific blocker probeand/or the first and/or second allele-specific primer comprises at leastone modified base. In some embodiments, the modified base(s) mayincrease the difference in the Tm between matched and mismatched targetsequences and/or decrease mismatch priming efficiency, thereby improvingnot only assay specificity bust also selectivity. Such modified base(s)may include, for example, 8-Aza-7-deaza-dA (ppA), 8-Aza-7-deaza-dG(ppG), 2′-Deoxypseudoisocytidine (iso dC), 5-fluoro-2′-deoxyuridine(fdU), locked nucleic acid (LNA), or 2′-O,4′-C-ethylene bridged nucleicacid (ENA) bases (see also, for example, FIG. 4B). In some embodimentsthe modified base is located at (a) the 3′-end, (b) the 5′-end, (c) atan internal position or at any combination of (a), (b) or (c) withinsaid first and/or second allele-specific blocker probe and/or the firstand/or second allele-specific primer.

In some embodiments of the kits, the first and/or second detector probesare the same. In some embodiments of the disclosed kits the first and/orsecond detector probes are different. In some embodiments of thedisclosed kits, the first and/or second detector probes aresequence-based or locus-specific detector probes. In other embodimentsthe first and/or second detector probe are 5′ nuclease probes. In someexemplary embodiments, the first and/or second detector probes comprisean MGB moiety, a reporter moiety (e.g., FAM™, TET™, JOE™, VIC™, or SYBR®Green), a quencher moiety (e.g., Black Hole Quencher™ or TAMRA™), and/ora passive reference (e.g., ROX™). In some exemplary embodiments, thefirst and/or second detector probe are designed according to the methodsand principles described in U.S. Pat. No. 6,727,356 (the disclosure ofwhich is incorporated herein by reference in its entirety). In someexemplary embodiments, the detector probe is a TaqMan® probe.

In some embodiments of the kits, the first locus-specific primer and thesecond locus-specific primer comprise the same sequence. In someembodiments the first locus-specific primer and the secondlocus-specific primer are the same sequence.

In some embodiments of the kits, the first and/or second reactionmixture can further comprise a polymerase; dNTPs; other reagents and/orbuffers suitable for PCR amplification; and/or a template sequence ornucleic acid sample. In some embodiments, the polymerase can be a DNApolymerase. In some other embodiments, the polymerase can bethermostable, such as Taq DNA polymerase.

In some embodiments, the compositions, methods and kits of the presentinvention provide high allelic discrimination specificity andselectivity. In some embodiments, the quantitative determination ofspecificity and/or selectivity comprises a comparison of Ct valuesbetween a first set of amplicons and a second set of amplicons. In someembodiments, selectivity is at a level whereby a single copy of a givenallele in about 1 million copies of another allele or alleles can bedetected.

The foregoing has described various embodiments of the invention thatprovide improved detection and discrimination of allelic variants usingone or more of the following: (a) tailed allele-specific primers; (b)low allele-specific primer concentration; (c) allele-specific primersdesigned to have lower Tms; (d) allele-specific primers designed totarget discriminating bases; (e) allele-specific blocker probescontaining MGB, designed to prevent amplification from alternative, andpotentially more abundant, allelic variants in a sample; and (f)allele-specific blocker probes and/or allele-specific primers designedto comprise modified bases in order to increase the delta Tm betweenmatched and mismatched target sequences.

While particular embodiments employing several of the above improvementshave been discussed herein, it will be apparent to the skilled artisanthat depending on the nature of the sample to be examined, variouscombinations of the above improvements can be combined to arrive at afavorable result. Thus, for example, non-MGB blocker probes can be usedwith an embodiment that include methods employing allele-specificprimers containing modified bases to increase delta Tm; such primers canalso be designed to target discriminating bases; and the primers can beused at low primer concentrations. Accordingly, alternative embodimentsbased upon the present disclosure can be used to achieve a suitablelevel of allelic detection.

The present disclosure provides the advantage that any of thecombinations of listed improvements could be utilized by a skilledartisan in a particular situation. For example, the current inventioncan include a method or reaction mixture that employs improvements a, c,d and f; improvements b, c, and e; or improvements

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several exemplary embodiments ofthe disclosure and together with the description, serve to explaincertain teachings.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings described beloware for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1 depicts a schematic of an illustrative embodiment of cast-PCR. Insome embodiments, components of cast-PCR include the following: onelocus-specific TaqMan probe (LST); two MGB blockers: oneallele-1-specific MGB blocker (MGB1) and one allele-2-specific MGBblocker (MGB2); 3 PCR primers: one locus-specific PCR primer (LSP); oneallele-1-specific primer (ASP1) and one allele-2-specific primer (ASP2).

FIG. 2 depicts a schematic of an illustrative embodiment of cast-PCRusing allele-specific blocker probes comprising highly discriminatingbases for detecting rare allelic variants. Highly discriminating basesmay include, for example, A/A, A/G, G/A, G/G, A/C, C/A. The leastdiscriminating bases may include, for example, C/C, T/C, G/T, T/G, C/T.In some embodiments, for example, for detection of A-G or C-T SNPs, A &G are used as the discriminating base if A/T is allelic variant (e.g.,mutant allele); or C & T are used as the discriminating base if C/Gallelic variant (e.g., mutant allele).

FIG. 3 depicts a schematic of an illustrative embodiment of cast-PCRusing an allele-specific blocker probe with an MGB moiety at the 5′ end.In some embodiments the blocker moiety at the 3′-end of the probe mayinclude, for example, NH₂, biotin, MGB, PO₄, and PEG.

FIG. 4A depicts a schematic of an illustrative embodiment of cast-PCRusing modified bases in an MGB blocker probe or allele-specific primer.(G* represents ppG.)

FIG. 4B depicts some examples of modified bases of an MGB blocker probeor allele-specific primer.

FIG. 5 depicts the TaqMan-like sensitivity and dynamic range of oneexemplary embodiment of cast-PCR.

FIG. 6 depicts the sequence of KRAS mutations at codons 12 and 13 thatare detectable using cast-PCR methods. KRAS mutations at codons 12 and13 are associated with resistance to cetuxima or panitumumab inmetastatic colorectal cancer (Di Nicolantonio F., et al., J Clin Oncol.2008; 26:5705-12). FIG. 6 discloses SEQ ID NO: 79.

FIG. 7 depicts the specificity of KRAS mutation detection using cast-PCRassays in one exemplary embodiment.

FIG. 8 depicts one exemplary embodiment using cast-PCR methods to detecta single copy of mutant DNA in 10⁶ copies of wild-type DNA.

FIG. 9 depicts detection of the relative copy number of mutant samples(KRAS-G12A) spiked in wild type samples using cast-PCR methods.

FIG. 10 depicts a number of different tumor markers (SNPs) detected intumor samples using one exemplary embodiment of cast-PCR.

FIG. 11A-E shows a list of exemplary primers and probes used in cast-PCRassays. Nucleotides shown in lower case are the tailed portion of theprimers. The nucleotide-portion of allele-specific primers (ASP) is atthe 3′-most terminus of each primer and are indicated in bold. Theallele positions of the blocker probes (MGB) are located at variousinternal positions relative to the blocker moieties, in some cases, areindicated in bold. FIG. 11A discloses ‘ASP1’ as SEQ ID NOS 80-84,respectively, in order of appearance, ‘LSP’ as SEQ ID NOS 85-89,respectively, in order of appearance, ‘MGB1’ as SEQ ID NOS 90-94,respectively, in order of appearance, ‘ASP2’ as SEQ ID NOS 95-99,respectively, in order of appearance, ‘LST’ as SEQ ID NOS 100-104,respectively, in order of appearance, and ‘MGB2’ as SEQ ID NOS 105-109,respectively, in order of appearance. FIG. 11B discloses ‘ASP1’ as SEQID NOS 110-135, respectively, in order of appearance, and ‘ASP2’ as SEQID NOS 136-161, respectively, in order of appearance. FIG. 11C discloses‘ASP1’ as SEQ ID NOS 162-187, respectively, in order of appearance, and‘ASP2’ as SEQ ID NOS 188-213, respectively, in order of appearance. FIG.11D discloses ‘LSP’ as SEQ ID NOS 214-239, respectively, in order ofappearance, and ‘LST’ as SEQ ID NOS 240-265, respectively, in order ofappearance. FIG. 11E discloses ‘MGB1’ as SEQ ID NOS 266-292,respectively, in order of appearance, ‘MGB2’ as SEQ ID NOS 293-317,respectively, in order of appearance.

FIG. 12 depicts, in one exemplary embodiment, the specificity of allelicdiscrimination for samples that were pre-amplified prior to analysis bycast-PCR.

FIG. 13 depicts, in on exemplary embodiment, the specificity of allelicdiscrimination for cast-PCR assays performed using tailed versusnon-tailed allele-specific primers.

FIG. 14 depicts, in on exemplary embodiment, the specificity of allelicdiscrimination for samples analyzed by cast-PCR versus samples analyzedby ASB-PCR methods.

FIG. 15 depicts, in on exemplary embodiment, the specificity of allelicdiscrimination for cast-PCR assays performed using MGB blocker probes orphosphate blocker probes.

FIG. 16 depicts, in one exemplary embodiment, the specificity of allelicdiscrimination for cast-PCR assays performed using LNA-modifiedallele-specific primers.

FIG. 17 compares, in one exemplary embodiment, the specificity ofallelic discrimination for cast-PCR assays performed using variouschemically-modified allele-specific primers.

FIGS. 18A and 18B show a list of exemplary allele-specific primers andprobes used in pre-amplification and cast-PCR assays. FIG. 18A discloses‘ASP1’ as SEQ ID NOS 318-324, respectively, in order of appearance,‘LSP’ as SEQ ID NOS 325-331, respectively, in order of appearance,‘ASP2’ as SEQ ID NOS 332-338, respectively, in order of appearance, and‘LST’ as SEQ ID NOS 339-345, respectively, in order of appearance. FIG.18B discloses ‘MGB1’ as SEQ ID NOS 346-352, respectively, in order ofappearance, and ‘MGB2’ as SEQ ID NOS 353-359, respectively, in order ofappearance.

FIG. 19A-D shows a list of exemplary primers and probes used in cast-PCRassays using either tailed (ASP +tail) or non-tailed (ASP −tail)allele-specific primers. (The tailed portion of the ASP +tail primersare indicated in bold.). FIG. 19A discloses ‘ASP1−tail’ as SEQ ID NOS360-371, respectively, in order of appearance, and ‘ASP2−tail’ as SEQ IDNOS 372-383, respectively, in order of appearance. FIG. 19B discloses‘ASP1+tail’ as SEQ ID NOS 384-395, respectively, in order of appearance,and ‘ASP2+tail’ as SEQ ID NOS 396-407, respectively, in order ofappearance. FIG. 19C discloses ‘LSP’ as SEQ ID NOS 408-419,respectively, in order of appearance, and ‘LST’ as SEQ ID NOS 420-431,respectively, in order of appearance. FIG. 19D discloses ‘MGB1’ as SEQID NOS 432-443, respectively, in order of appearance, and ‘MGB2’ as SEQID NOS 444-455, respectively, in order of appearance.

FIG. 20A-C shows a list of exemplary primers and probes used in ASB-PCR.The blocker probes used in ASB-PCR comprise a phosphate group at the3′-end of the blocker probes (PHOS). FIG. 20A discloses ‘ASP1’ as SEQ IDNOS 456-467, respectively, in order of appearance, and ‘ASP2’ as SEQ IDNOS 468-479, respectively, in order of appearance. FIG. 20B discloses‘LSP’ as SEQ ID NOS 480-491, respectively, in order of appearance, and‘LST’ as SEQ ID NOS 492-503, respectively, in order of appearance. FIG.20C discloses ‘PHOS1’ as SEQ ID NOS 504-515, respectively, in order ofappearance, and ‘PHOS2’ as SEQ ID NOS 516-527, respectively, in order ofappearance.

FIG. 21A-C shows a list of exemplary primers and probes used in cast-PCRassays performed using LNA-modified allele-specific primers. In thisexemplary embodiment, the LNA modifications of the ASP are at the3′-ends. (“+” indicates the LNA modified nucleotide and are notated inparentheses.) FIG. 21A discloses ‘ASP1’ as SEQ ID NOS 528-539,respectively, in order of appearance, and ‘ASP2’ as SEQ ID NOS 540-551,respectively, in order of appearance. FIG. 21B discloses ‘LSP’ as SEQ IDNOS 552-563, and ‘LST’ as SEQ ID NOS 564-575, respectively, in order ofappearance. FIG. 21C discloses ‘MGB1’ as SEQ ID NOS 576-587,respectively, in order of appearance, and ‘MGB2’ as SEQ ID NOS 588-599,respectively, in order of appearance.

FIG. 22 shows a list of exemplary primers and probes used in cast-PCRassays performed using chemically modified allele-specific primers. Inthis exemplary embodiment, the chemical modifications (e.g., ppA, ppG,fdU, and iso dC) of the ASP are at the 3′-ends. (The chemically modifiednucleotides are shown in parentheses.) FIG. 22 discloses ‘ASP1’ as SEQID NOS 600-605, respectively, in order of appearance, ‘LSP’ as SEQ IDNOS 606-611, respectively, in order of appearance, ‘MGB1’ as SEQ ID NOS612-617, respectively, in order of appearance, ‘ASP2’ as SEQ ID NOS618-623, respectively, in order of appearance, ‘LST’ as SEQ ID NOS624-629, respectively, in order of appearance, and ‘MGB2’ as SEQ ID NOS630-635, respectively, in order of appearance.

DETAILED DESCRIPTION I. Introduction

The selective amplification of an allele of interest is oftencomplicated by factors including the mispriming and extension of amismatched allele-specific primer on an alternative allele. Suchmispriming and extension can be especially problematic in the detectionof rare alleles present in a sample populated by an excess of anotherallelic variant. When in sufficient excess, the mispriming and extensionof the other allelic variant may obscure the detection of the allele ofinterest. When using PCR-based methods, the discrimination of aparticular allele in a sample containing alternative allelic variantsrelies on the selective amplification of an allele of interest, whileminimizing or preventing amplification of other alleles present in thesample.

A number of factors have been identified, which alone or in combination,contribute to the enhanced discriminating power of allele-specific PCR.As disclosed herein, a factor which provides a greater ΔCt value betweena mismatched and matched allele-specific primer is indicative of greaterdiscriminating power between allelic variants. Such factors found toimprove discrimination of allelic variants using the present methodsinclude, for example, the use of one or more of the following: (a)tailed allele-specific primers; (b) low allele-specific primerconcentration; (c) allele-specific primers designed to have lower Tms;(d) allele-specific primers designed to target discriminating bases; (e)allele-specific blocker probes designed to prevent amplification fromalternative, and potentially more abundant, allelic variants in asample; and (f) allele-specific blocker probes and/or allele-specificprimers designed to comprise modified bases in order to increase thedelta Tm between matched and mismatched target sequences.

The above-mentioned factors, especially when used in combination, caninfluence the ability of allele-specific PCR to discriminate betweendifferent alleles present in a sample. Thus, the present disclosurerelates generally to novel amplification methods referred to ascast-PCR, which utilizes a combination of factors referred to above toimprove discrimination of allelic variants during PCR by increasing ΔCtvalues. In some embodiments, the present methods can involve high levelsof selectivity, wherein one mutant molecule in a background of at least1,000 to 1,000,000, such as about 1000-10,000, about 10,000 to 100,000,or about 100,000 to 1,000,000 wild type molecules, or any fractionalranges in between can be detected. In some embodiments, the comparisonof a first set of amplicons and a second set of amplicons involving thedisclosed methods provides improvements in specificity from 1,000× to100,000,000× fold difference, such as about 1000-10,000×, about 10,000to 100,000×, about 100,000 to 1,000,000× or about 1,000,000 to100,000,000× fold difference, or any fractional ranges in between.

II. Definitions

For the purposes of interpreting this specification, the followingdefinitions will apply and whenever appropriate, terms used in thesingular will also include the plural and vice versa. In the event thatany definition set forth below conflicts with the usage of that word inany other document, including any document incorporated herein byreference, the definition set forth below shall always control forpurposes of interpreting this specification and its associated claimsunless a contrary meaning is clearly intended.

As used herein, the term “allele” refers generally to alternative DNAsequences at the same physical locus on a segment of DNA, such as, forexample, on homologous chromosomes. An allele can refer to DNA sequenceswhich differ between the same physical locus found on homologouschromosomes within a single cell or organism or which differ at the samephysical locus in multiple cells or organisms (“allelelic variant”). Insome instances, an allele can correspond to a single nucleotidedifference at a particular physical locus. In other embodiments andallele can correspond to nucleotide (single or multiple) insertion ordeletion.

As used herein, the term “allele-specific primer” refers to anoligonucleotide sequence that hybridizes to a sequence comprising anallele of interest, and which when used in PCR can be extended toeffectuate first strand cDNA synthesis. Allele-specific primers arespecific for a particular allele of a given target DNA or loci and canbe designed to detect a difference of as little as one nucleotide in thetarget sequence. Allele-specific primers may comprise an allele-specificnucleotide portion, a target-specific portion, and/or a tail.

As used herein, the terms “allele-specific nucleotide portion” or“allele-specific target nucleotide” refers to a nucleotide ornucleotides in an allele-specific primer that can selectively hybridizeand be extended from one allele (for example, a minor or mutant allele)at a given locus to the exclusion of the other (for example, thecorresponding major or wild type allele) at the same locus.

As used herein, the term “target-specific portion” refers to the regionof an allele-specific primer that hybridizes to a target polynucleotidesequence. In some embodiments, the target-specific portion of theallele-specific primer is the priming segment that is complementary tothe target sequence at a priming region 5′ of the allelic variant to bedetected. The target-specific portion of the allele-specific primer maycomprise the allele-specific nucleotide portion. In other instances, thetarget-specific portion of the allele-specific primer is adjacent to the3′ allele-specific nucleotide portion.

As used herein, the terms “tail” or “5′-tail” refers to the non-3′ endof a primer. This region typically will, although does not have tocontain a sequence that is not complementary to the targetpolynucleotide sequence to be analyzed. The 5′ tail can be any of about2-30, 2-5, 4-6, 5-8, 6-12, 7-15, 10-20, 15-25 or 20-30 nucleotides, orany range in between, in length.

As used herein, the term “allele-specific blocker probe” (also referredto herein as “blocker probe,” “blocker,”) refers to an oligonucleotidesequence that binds to a strand of DNA comprising a particular allelicvariant which is located on the same, opposite or complementary strandas that bound by an allelic-specific primer, and reduces or preventsamplification of that particular allelic variant. As discussed ingreater detail herein, allele-specific blocker probes generally comprisemodifications, e.g., at the 3′-OH of the ribose ring, which preventprimer extension by a polymerase. The allele-specific blocker probe canbe designed to anneal to the same or opposing strand of what theallele-specific primer anneals to and can be modified with a blockinggroup (e.g., a “non-extendable blocker moiety”) at its 3′ terminal end.Thus, a blocker probe can be designed, for example, so as to tightlybind to a wild type allele (e.g., abundant allelic variant) in order tosuppress amplification of the wild type allele while amplification isallowed to occur on the same or opposing strand comprising a mutantallele (e.g., rare allelic variant) by extension of an allele-specificprimer. In illustrative examples, the allele-specific blocker probes donot include a label, such as a fluorescent, radioactive, orchemiluminescent label

As used herein, the term “non-extendable blocker moiety” refersgenerally to a modification on an oligonucleotide sequence such as aprobe and/or primer which renders it incapable of extension by apolymerase, for example, when hybridized to its complementary sequencein a PCR reaction. Common examples of blocker moieties includemodifications of the ribose ring 3′-OH of the oligonucleotide, whichprevents addition of further bases to the ‘3-end of the oligonucleotidesequence a polymerase. Such 3’-OH modifications are well known in theart. (See, e.g., Josefsen, M., et al., Molecular and Cellular Probes, 23(2009):201-223; McKinzie, P. et al., Mutagenesis. 2006, 21(6):391-7;Parsons, B. et al., Methods Mol Biol. 2005, 291:235-45; Parsons, B. etal., Nucleic Acids Res. 1992, 25:20(10):2493-6; and Morlan, J. et al.,PLoS One 2009, 4 (2): e4584, the disclosures of which are incorporatedherein by reference in their entireties.)

As used herein, the terms “MGB,” “MGB group,” “MGB compound,” or “MBGmoiety” refers to a minor groove binder. When conjugated to the 3′ endof an oligonucleotide, an MGB group can function as a non-extendableblocker moiety.

An MGB is a molecule that binds within the minor groove of doublestranded DNA. Although a general chemical formula for all known MGBcompounds cannot be provided because such compounds have widely varyingchemical structures, compounds which are capable of binding in the minorgroove of DNA, generally speaking, have a crescent shape threedimensional structure. Most MGB moieties have a strong preference forA-T (adenine and thymine) rich regions of the B form of double strandedDNA. Nevertheless, MGB compounds which would show preference to C-G(cytosine and guanine) rich regions are also theoretically possible.Therefore, oligonucleotides comprising a radical or moiety derived fromminor groove binder molecules having preference for C-G regions are alsowithin the scope of the present invention.

Some MGBs are capable of binding within the minor groove of doublestranded DNA with an association constant of 10³M⁻¹ or greater. Thistype of binding can be detected by well-established spectrophotometricmethods such as ultraviolet (UV) and nuclear magnetic resonance (NMR)spectroscopy and also by gel electrophoresis. Shifts in UV spectra uponbinding of a minor groove binder molecule and NMR spectroscopy utilizingthe “Nuclear Overhauser” (NOSEY) effect are particularly well known anduseful techniques for this purpose. Gel electrophoresis detects bindingof an MGB to double stranded DNA or fragment thereof, because upon suchbinding the mobility of the double stranded DNA changes.

A variety of suitable minor groove binders have been described in theliterature. See, for example, Kutyavin, et al. U.S. Pat. No. 5,801,155;Wemmer, D. E., and Dervan P. B., Current Opinion in Structural Biology,7:355-361 (1997); Walker, W. L., Kopka, J. L. and Goodsell, D. S.,Biopolymers, 44:323-334 (1997); Zimmer, C.& Wahnert, U. Prog. Biophys.Molec. Bio. 47:31-112 (1986) and Reddy, B. S. P., Dondhi, S. M., andLown, J. W., Pharmacol. Therap., 84:1-111 (1999) (the disclosures ofwhich are herein incorporated by reference in their entireties). Apreferred MGB in accordance with the present disclosure is DPI₃.Synthesis methods and/or sources for such MGBs are also well known inthe art. (See, e.g., U.S. Pat. Nos. 5,801,155; 6,492,346; 6,084,102; and6,727,356, the disclosures of which are incorporated herein by referencein their entireties.)

As used herein, the term “MGB blocker probe,” “MBG blocker,” or “MGBprobe” is an oligonucleotide sequence and/or probe further attached to aminor groove binder moiety at its 3′ and/or 5′ end. Oligonucleotidesconjugated to MGB moieties form extremely stable duplexes withsingle-stranded and double-stranded DNA targets, thus allowing shorterprobes to be used for hybridization based assays. In comparison tounmodified DNA, MGB probes have higher melting temperatures (Tm) andincreased specificity, especially when a mismatch is near the MGB regionof the hybridized duplex. (See, e.g., Kutyavin, I. V., et al., NucleicAcids Research, 2000, Vol. 28, No. 2: 655-661).

As used herein, the term “modified base” refers generally to anymodification of a base or the chemical linkage of a base in a nucleicacid that differs in structure from that found in a naturally occurringnucleic acid. Such modifications can include changes in the chemicalstructures of bases or in the chemical linkage of a base in a nucleicacid, or in the backbone structure of the nucleic acid. (See, e.g.,Latorra, D. et al., Hum Mut 2003, 2:79-85. Nakiandwe, J. et al., PlantMethod 2007, 3:2.)

As used herein, the term “detector probe” refers to any of a variety ofsignaling molecules indicative of amplification. For example, SYBR®Green and other DNA-binding dyes are detector probes. Some detectorprobes can be sequence-based (also referred to herein as “locus-specificdetector probe”), for example 5′ nuclease probes. Various detectorprobes are known in the art, for example (TaqMan® probes describedherein (See also U.S. Pat. No. 5,538,848) various stem-loop molecularbeacons (See, e.g., U.S. Pat. Nos. 6,103,476 and 5,925,517 and Tyagi andKramer, 1996, Nature Biotechnology 14:303-308), stemless or linearbeacons (See, e.g., WO 99/21881), PNA Molecular Beacons™ (See, e.g.,U.S. Pat. Nos. 6,355,421 and 6,593,091), linear PNA beacons (See, e.g.,Kubista et al., 2001, SPIE 4264:53-58), non-FRET probes (See, e.g., U.S.Pat. No. 6,150,097), Sunrise®/Amplifluor® probes (U.S. Pat. No.6,548,250), stem-loop and duplex Scorpion™ probes (Solinas et al., 2001,Nucleic Acids Research 29:E96 and U.S. Pat. No. 6,589,743), bulge loopprobes (U.S. Pat. No. 6,590,091), pseudo knot probes (U.S. Pat. No.6,589,250), cyclicons (U.S. Pat. No. 6,383,752), MGB Eclipse™ probe(Epoch Biosciences), hairpin probes (U.S. Pat. No. 6,596,490), peptidenucleic acid (PNA) light-up probes, self-assembled nanoparticle probes,and ferrocene-modified probes described, for example, in U.S. Pat. No.6,485,901; Mhlanga et al., 2001, Methods 25:463-471; Whitcombe et al.,1999, Nature Biotechnology. 17:804-807; Isacsson et al., 2000, MolecularCell Probes. 14:321-328; Svanvik et al., 2000, Anal Biochem. 281:26-35;Wolffs et al., 2001, Biotechniques 766:769-771; Tsourkas et al., 2002,Nucleic Acids Research. 30:4208-4215; Riccelli et al., 2002, NucleicAcids Research 30:4088-4093; Zhang et al., 2002 Shanghai. 34:329-332;Maxwell et al., 2002, J. Am. Chem. Soc. 124:9606-9612; Broude et al.,2002, Trends Biotechnol. 20:249-56; Huang et al., 2002, Chem Res.Toxicol. 15:118-126; and Yu et al., 2001, J. Am. Chem. Soc14:11155-11161. Detector probes can comprise reporter dyes such as, forexample, 6-carboxyfluorescein (6-FAM) or tetrachlorofluorescin (TET).Detector probes can also comprise quencher moieties such astetramethylrhodamine (TAMRA), Black Hole Quenchers (Biosearch), IowaBlack (IDT), QSY quencher (Molecular Probes), and Dabsyl and Dabcelsulfonate/carboxylate Quenchers (Epoch). Detector probes can alsocomprise two probes, wherein for example a fluor is on one probe, and aquencher on the other, wherein hybridization of the two probes togetheron a target quenches the signal, or wherein hybridization on a targetalters the signal signature via a change in fluorescence. Detectorprobes can also comprise sulfonate derivatives of fluorescein dyes withSO₃ instead of the carboxylate group, phosphoramidite forms offluorescein, phosphoramidite forms of CY5 (available, for example, fromAmersham Biosciences-GE Healthcare).

As used herein, the term “locus-specific primer” refers to anoligonucleotide sequence that hybridizes to products derived from theextension of a first primer (such as an allele-specific primer) in a PCRreaction, and which can effectuate second strand cDNA synthesis of saidproduct. Accordingly, in some embodiments, the allele-specific primerserves as a forward PCR primer and the locus-specific primer serves as areverse PCR primer, or vice versa. In some preferred embodiments,locus-specific primers are present at a higher concentration as comparedto the allele-specific primers.

As used herein, the term “rare allelic variant” refers to a targetpolynucleotide present at a lower level in a sample as compared to analternative allelic variant. The rare allelic variant may also bereferred to as a “minor allelic variant” and/or a “mutant allelicvariant.” For instance, the rare allelic variant may be found at afrequency less than 1/10, 1/100, 1/1,000, 1/10,000, 1/100,000,1/1,000,000, 1/10,000,000, 1/100,000,000 or 1/1,000,000,000 compared toanother allelic variant for a given SNP or gene. Alternatively, the rareallelic variant can be, for example, less than 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 50, 75, 100, 250, 500, 750, 1,000, 2,500, 5,000, 7,500,10,000, 25,000, 50,000, 75,000, 100,000, 250,000, 500,000, 750,000, or1,000,000 copies per 1, 10, 100, 1,000 micro liters of a sample or areaction volume.

As used herein, the terms “abundant allelic variant” may refer to atarget polynucleotide present at a higher level in a sample as comparedto an alternative allelic variant. The abundant allelic variant may alsobe referred to as a “major allelic variant” and/or a “wild type allelicvariant.” For instance, the abundant allelic variant may be found at afrequency greater than 10×, 100×, 1,000×, 10,000×, 100,000×, 1,000,000×,10,000,000×, 100,000,000× or 1,000,000,000× compared to another allelicvariant for a given SNP or gene. Alternatively, the abundant allelicvariant can be, for example, greater than 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 50, 75, 100, 250, 500, 750, 1,000, 2,500, 5,000, 7,500,10,000, 25,000, 50,000, 75,000, 100,000, 250,000, 500,000, 750,000,1,000,000 copies per 1, 10, 100, 1,000 micro liters of a sample or areaction volume.

As used herein, the terms “first” and “second” are used to distinguishthe components of a first reaction (e.g., a “first” reaction; a “first”allele-specific primer) and a second reaction (e.g., a “second”reaction; a “second” allele-specific primer). By convention, as usedherein the first reaction amplifies a first (for example, a rare)allelic variant and the second reaction amplifies a second (for example,an abundant) allelic variant or vice versa.

As used herein, both “first allelic variant” and “second allelicvariant” can pertain to alleles of a given locus from the same organism.For example, as might be the case in human samples (e.g., cells)comprising wild type alleles, some of which have been mutated to form aminor or rare allele. The first and second allelic variants of thepresent teachings can also refer to alleles from different organisms.For example, the first allele can be an allele of a genetically modifiedorganism, and the second allele can be the corresponding allele of awild type organism. The first allelic variants and second allelicvariants of the present teachings can be contained on gDNA, as well asmRNA and cDNA, and generally any target nucleic acids that exhibitsequence variability due to, for example, SNP or nucleotide(s) insertionand/or deletion mutations.

As used herein, the term “thermostable” or “thermostable polymerase”refers to an enzyme that is heat stable or heat resistant and catalyzespolymerization of deoxyribonucleotides to form primer extension productsthat are complementary to a nucleic acid strand. Thermostable DNApolymerases useful herein are not irreversibly inactivated whensubjected to elevated temperatures for the time necessary to effectdestabilization of single-stranded nucleic acids or denaturation ofdouble-stranded nucleic acids during PCR amplification. Irreversibledenaturation of the enzyme refers to substantial loss of enzymeactivity. Preferably a thermostable DNA polymerase will not irreversiblydenature at about 90°-100° C. under conditions such as is typicallyrequired for PCR amplification.

As used herein, the term “PCR amplifying” or “PCR amplification” refersgenerally to cycling polymerase-mediated exponential amplification ofnucleic acids employing primers that hybridize to complementary strands,as described for example in Innis et al., PCR Protocols: A Guide toMethods and Applications, Academic Press (1990). Devices have beendeveloped that can perform thermal cycling reactions with compositionscontaining fluorescent indicators which are able to emit a light beam ofa specified wavelength, read the intensity of the fluorescent dye, anddisplay the intensity of fluorescence after each cycle. Devicescomprising a thermal cycler, light beam emitter, and a fluorescentsignal detector, have been described, e.g., in U.S. Pat. Nos. 5,928,907;6,015,674; 6,174,670; and 6,814,934 and include, but are not limited to,the ABI Prism® 7700 Sequence Detection System (Applied Biosystems,Foster City, Calif.), the ABI GeneAmp® 5700 Sequence Detection System(Applied Biosystems, Foster City, Calif.), the ABI GeneAmp® 7300Sequence Detection System (Applied Biosystems, Foster City, Calif.), theABI GeneAmp® 7500 Sequence Detection System (Applied Biosystems, FosterCity, Calif.), the StepOne™ Real-Time PCR System (Applied Biosystems,Foster City, Calif.) and the ABI GeneAmp® 7900 Sequence Detection System(Applied Biosystems, Foster City, Calif.).

As used herein, the term “Tm” or “melting temperature” of anoligonucleotide refers to the temperature (in degrees Celsius) at which50% of the molecules in a population of a single-strandedoligonucleotide are hybridized to their complementary sequence and 50%of the molecules in the population are not-hybridized to saidcomplementary sequence. The Tm of a primer or probe can be determinedempirically by means of a melting curve. In some cases it can also becalculated using formulas well known in the art (See, e.g., Maniatis,T., et al., Molecular cloning: a laboratory manual/Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.: 1982).

As used herein, the term “sensitivity” refers to the minimum amount(number of copies or mass) of a template that can be detected by a givenassay. As used herein, the term “specificity” refers to the ability ofan assay to distinguish between amplification from a matched templateversus a mismatched template. Frequently, specificity is expressed asΔC_(t)=Ct_(mismatch)−Ct_(match). An improvement in specificity or“specificity improvement” or “fold difference” is expressed herein as2^((ΔCt) ^(_) ^(condion1−(ΔCt) ^(_) ^(condition2)). The term“selectivity” refers to the extent to which an AS-PCR assay can be usedto determine minor (often mutant) alleles in mixtures withoutinterferences from major (often wild type) alleles. Selectivity is oftenexpressed as a ratio or percentage. For example, an assay that candetect 1 mutant template in the presence of 100 wild type templates issaid to have a selectivity of 1:100 or 1%. As used herein, assayselectivity can also be calculated as ½^(ΔCt) or as a percentage using(½^(ΔCt)×100).

As used herein, the term “Ct” or “Ct value” refers to threshold cycleand signifies the cycle of a PCR amplification assay in which signalfrom a reporter that is indicative of amplicon generation (e.g.,fluorescence) first becomes detectable above a background level. In someembodiments, the threshold cycle or “Ct” is the cycle number at whichPCR amplification becomes exponential.

As used herein, the term “delta Ct” or “ΔCt” refers to the difference inthe numerical cycle number at which the signal passes the fixedthreshold between two different samples or reactions. In someembodiments delta Ct is the difference in numerical cycle number atwhich exponential amplification is reached between two different samplesor reactions. The delta Ct can be used to identify the specificitybetween a matched primer to the corresponding target nucleic acidsequence and a mismatched primer to the same corresponding targetnucleic acid sequence.

In some embodiments, the calculation of the delta Ct value between amismatched primer and a matched primer is used as one measure of thediscriminating power of allele-specific PCR. In general, any factorwhich increases the difference between the Ct value for an amplificationreaction using a primer that is matched to a target sequence (e.g., asequence comprising an allelic variant of interest) and that of amismatched primer will result in greater allele discrimination power.

According to various embodiments, a Ct value may be determined using aderivative of a PCR curve. For example, a first, second, or nth orderderivative method may be performed on a PCR curve in order to determinea Ct value. In various embodiments, a characteristic of a derivative maybe used in the determination of a Ct value. Such characteristics mayinclude, but are not limited by, a positive inflection of a secondderivative, a negative inflection of a second derivative, a zerocrossing of the second derivative, or a positive inflection of a firstderivative. In various embodiments, a Ct value may be determined using athresholding and baselining method. For example, an upper bound to anexponential phase of a PCR curve may be established using a derivativemethod, while a baseline for a PCR curve may be determined to establisha lower bound to an exponential phase of a PCR curve. From the upper andlower bound of a PCR curve, a threshold value may be established fromwhich a Ct value is determined. Other methods for the determination of aCt value known in the art, for example, but not limited by, variousembodiments of a fit point method, and various embodiments of asigmoidal method (See, e.g., U.S. Pat. Nos. 6,303,305; 6,503,720;6,783,934, 7,228,237 and U.S. Application No. 2004/0096819; thedisclosures of which are herein incorporated by reference in theirentireties).

III. Compositions, Methods and Kits

In one aspect, the present invention provides compositions for use inidentifying and/or quantitating an allelic variant in a nucleic acidsample. Some of these compositions can comprise: (a) an allele-specificprimer; (b) an allele-specific blocker probe; (c) a detector probe; and(d) a locus-specific primer, or any combinations thereof. In someembodiments of the compositions, the compositions may further comprise apolymerase, dNTPs, reagents and/or buffers suitable for PCRamplification, and/or a template sequence or nucleic acid sample. Insome embodiments, the polymerase can be thermostable.

In another aspect, the invention provides compositions comprising: (i) afirst allele-specific primer, wherein an allele-specific nucleotideportion of the first allele-specific primer is complementary to thefirst allelic variant of a target sequence; and (ii) a firstallele-specific blocker probe that is complementary to a region of thetarget sequence comprising the second allelic variant, wherein saidregion encompasses a position corresponding to the binding position ofthe allele-specific nucleotide portion of the first allele-specificprimer, and wherein the first allele-specific blocker probe comprises aminor groove binder.

In some illustrative embodiments, the compositions can further include alocus-specific primer that is complementary to a region of the targetsequence that is 3′ from the first allelic variant and on the oppositestrand.

In further embodiments, the compositions can further include a detectorprobe.

In another aspect, the present invention provides methods for amplifyingan allele-specific sequence. Some of these methods can include: (a)hybridizing an allele-specific primer to a first nucleic acid moleculecomprising a target allele; (b) hybridizing an allele-specific blockerprobe to a second nucleic acid molecule comprising an alternative allelewherein the alternative allele corresponds to the same loci as thetarget allele; (c) hybridizing a locus-specific detector probe to thefirst nucleic acid molecule; (d) hybridizing a locus-specific primer tothe extension product of the allele-specific primer and (e) PCRamplifying the target allele.

In another aspect, the present invention provides methods for detectingand/or quantitating an allelic variant in a mixed sample. Some of thesemethods can involve: (a) in a first reaction mixture hybridizing a firstallele-specific primer to a first nucleic acid molecule comprising afirst allele (allele-1) and in a second reaction mixture hybridizing asecond allele-specific primer to a first nucleic acid moleculecomprising a second allele (allele-2), wherein the allele-2 correspondsto the same loci as allele-1; (b) in the first reaction mixturehybridizing a first allele-specific blocker probe to a second nucleicacid molecule comprising allele-2 and in the second reaction mixturehybridizing a second allele-specific blocker probe to a second nucleicacid molecule comprising allele-1; (c) in the first reaction mixture,hybridizing a first detector probe to the first nucleic acid moleculeand in the second reaction mixture, hybridizing a second detector probeto the first nucleic acid molecule; (d) in the first reaction mixturehybridizing a first locus-specific primer to the extension product ofthe first allele-specific primer and in the second reaction mixturehybridizing a second locus-specific primer to the extension product ofthe second allele-specific primer; and (e) PCR amplifying the firstnucleic acid molecule to form a first set or sample of amplicons and PCRamplifying the second nucleic acid molecule to form a second set orsample of amplicons; and (f) comparing the first set of amplicons to thesecond set of amplicons to quantitate allele-1 in the sample comprisingallele-2 and/or allele-2 in the sample comprising allele-1.

In yet another aspect, the present invention provides methods fordetecting and/or quantitating allelic variants. Some of these methodscan comprise: (a) PCR amplifying a first allelic variant in a firstreaction comprising (i) a low-concentration first allele-specificprimer, (ii) a first locus-specific primer, and (iii) a first blockerprobe to form first amplicons; (b) PCR amplifying a second allelicvariant in a second reaction comprising (i) a low-concentration secondallele-specific primer, (ii) a second locus-specific primer, and (iii) asecond blocker probe to form second amplicons; and (d) comparing thefirst amplicons to the second amplicons to quantitate the first allelicvariant in the sample comprising second allelic variants.

In yet another aspect, the present invention provides methods fordetecting a first allelic variant of a target sequence in a nucleic acidsample suspected of comprising at least a second allelic variant of thetarget sequence. Methods of this aspect include forming a first reactionmixture by combining the following: (i) a nucleic acid sample; (ii) afirst allele-specific primer, wherein an allele-specific nucleotideportion of the first allele-specific primer is complementary to thefirst allelic variant of the target sequence; (iii) a firstallele-specific blocker probe that is complementary to a region of thetarget sequence comprising the second allelic variant, wherein saidregion encompasses a position corresponding to the binding position ofthe allele-specific nucleotide portion of the first allele-specificprimer, and wherein the first allele-specific blocker probe comprises aminor groove binder; (iv) a first locus-specific primer that iscomplementary to a region of the target sequence that is 3′ from thefirst allelic variant and on the opposite strand; and (v) a firstdetector probe.

Next an amplification reaction, typically a PCR amplification reaction,is carried out on the first reaction mixture using the firstlocus-specific primer and the first allele-specific primer to form afirst amplicon. Then, the first amplicon is detected by a change in adetectable property of the first detector probe upon binding to theamplicon, thereby detecting the first allelic variant of the target genein the nucleic acid sample. The detector probe in some illustrativeembodiments is a 5′ nuclease probe. The detectable property in certainillustrative embodiments is fluorescence.

In some embodiments, the 3′ nucleotide position of the 5′ target regionof the first allele-specific primer is an allele-specific nucleotideposition. In certain other illustrative embodiments, including thoseembodiments where the 3′ nucleotide position of the 5′ target region ofthe first allele-specific primer is an allele-specific nucleotideposition, the blocking region of the allele-specific primer encompassesthe allele-specific nucleotide position. Furthermore, in illustrativeembodiments, the first allele-specific blocker probe includes a minorgroove binder. Furthermore, the allele-specific blocker probe in certainillustrative embodiments does not have a label, for example afluorescent label, or a quencher.

In certain illustrative embodiments, the quantity of the first allelicvariant is determined by evaluating the change in a detectable propertyof the first detector probe.

In certain illustrative embodiments, the method further includes forminga second reaction mixture by combining (i) the nucleic acid sample; (ii)a second allele-specific primer, wherein an allele-specific nucleotideportion of the second allele-specific primer is complementary to thesecond allelic variant of the target sequence; (iii) a secondallele-specific blocker probe that is complementary to a region of thetarget sequence comprising the first allelic variant, wherein saidregion encompasses a position corresponding to the binding position ofthe allele-specific nucleotide portion of the second allele-specificprimer, and wherein the second allele-specific blocker probe comprises aminor groove binder; (iv) a second locus-specific primer that iscomplementary to a region of the target sequence that is 3′ from thesecond allelic variant and on the opposite strand; and (v) a seconddetector probe. Next, an amplification reaction is carried out on thesecond reaction mixture using the second allele-specific primer and thelocus-specific primer, to form a second amplicon. Then the secondamplicon is detected by a change in a detectable property of thedetector probe.

In certain embodiments, the method further includes comparing the changein a detectable property of the first detector probe in the firstreaction mixture to the change in a detectable property of the seconddetector probe in the second reaction mixture.

In preferred embodiments, the methods further include a 2-stage cyclingprotocol. In some embodiments, the cycling protocol comprises a firststage of amplification that employs an initial number of cycles at alower annealing/extension temperature, followed by a second stage ofamplification that employs a number of cycles at a higherannealing/extension temperature. Due to the lower Tm of cast-PCRallele-specific primers (e.g., 53-56° C.), PCR is not optimal atstandard annealing/extension conditions (e.g., 60-64° C.). Consequently,lower annealing/extension temperatures are used during the initialcycling stage which improves cast-PCR efficiency significantly.

In some embodiments, the number of cycles used in the first stage of thecast-PCR cycling protocol is fewer than the number of cycles used in thesecond stage. In some embodiments of the cast-PCR methods, the number ofcycles used in the first stage of the cycling protocol is about 2%-20%,4%-18%, 6%-16%, 8%-14%, 10%-12%, or any percent in between, of the totalnumber of cycles used in the second stage. In some embodiments, thefirst stage employs between 1 to 10 cycles, 2 to 8 cycles, 3 to 7cycles, or 4 to 6 cycles, and all number of cycles in between, e.g., 2,3, 4, 5, 6, or 7 cycles.

In some embodiments, the number of cycles used in the second stage ofthe cast-PCR cycling protocol is greater than the number of cycles usedin the second stage. In some embodiments of the cast-PCR methods, thenumber of cycles used in the second stage of the cycling protocol is 5times, 6 times, 8 times, 10 times, 12 times, 18 times, 25 times, or 30times the number of cycles used in the first stage. In some embodiments,the second stage employs between 30 to 50 cycles, 35 to 48 cycles, 40 to46 cycles, or any number of cycles in between, e.g., 42, 43, 44, 45, or46 cycles.

In some embodiments, the lower annealing/extension temperature usedduring the first cycling stage is about 1° C., about 2° C., about 3° C.,about 4° C., or about 5° C. lower than the annealing/extensiontemperature used during the second cycling stage. In some preferredembodiments, the annealing/extension temperature of the first stage isbetween 50° C. to 60° C., 52° C. to 58° C., or 54° C. to 56° C., e.g.,53° C., 54° C., 55° C. or 55° C. In some preferred embodiments theannealing/extension temperature of the second stage is between 56° C. to66° C., 58° C. to 64° C., or 60° C. to 62° C., e.g., 58° C., 60° C., 62°C. or 64° C.

There are several major advantages of this 2-stage PCR cycling protocolused in cast-PCR that make it better than conventional AS-PCR methods.First, it improves the detection sensitivity by lowering the Ct valuefor matched targets or alleles. Next, it improves the specificity ofcast-PCR by increasing the ΔCt between Ct values of matched andmismatched sequences. Finally, it can improve the uniformity of cast-PCRby making it equally efficient across various assays.

In yet another aspect, the present invention provides a reaction mixturethat includes the following (i) nucleic acid molecule; (ii) anallele-specific primer, wherein an allele-specific nucleotide portion ofthe allele-specific primer is complementary to a first allelic variantof a target sequence; (iii) an allele-specific blocker probe that iscomplementary to a region of the target sequence comprising a secondallelic variant, wherein said region encompasses a positioncorresponding to the binding position of the allele-specific nucleotideportion of the allele-specific primer, and wherein the allele-specificblocker probe comprises a minor groove binder; (iv) a locus-specificprimer that is complementary to a region of the target sequence that is3′ from the first allelic variant and on the opposite strand; and (v) adetector probe.

In certain embodiments, the methods of the invention are used to detecta first allelic variant that is present at a frequency less than 1/10,1/100, 1/1,000, 1/10,000, 1/100,000, 1/1,000,000, 1/10,000,000,1/100,000,000 or 1/1,000,000,000, and any fractional ranges in between,of a second allelic variant for a given SNP or gene. In otherembodiments, the methods are used to detect a first allelic variant thatis present in less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75,100, 250, 500, 750, 1,000, 2,500, 5,000, 7,500, 10,000, 25,000, 50,000,75,000, 100,000, 250,000, 500,000, 750,000, 1,000,000 copies per 1, 10,100, 1,000 micro liters, and any fractional ranges in between, of asample or a reaction volume.

In some embodiments, the first allelic variant is a mutant. In someembodiments the second allelic variant is wild type. In someembodiments, the present methods can involve detecting one mutantmolecule in a background of at least 1,000 to 1,000,000, such as about1000 to 10,000, about 10,000 to 100,000, or about 100,000 to 1,000,000wild type molecules, or any fractional ranges in between. In someembodiments, the methods can provide high sensitivity and the efficiencyat least comparable to that of TaqMan®-based assays.

In some embodiments, the comparison of the first amplicons and thesecond amplicons involving the disclosed methods provides improvementsin specificity from 1,000× to 1,000,000× fold difference, such as about1000 to 10,000×, about 10,000 to 100,000×, or about 100,000 to1,000,000× fold difference, or any fractional ranges in between. In someembodiments, the size of the amplicons range from about 60-120nucleotides long.

In another aspect, the present invention provides kits for quantitatinga first allelic variant in a sample comprising an alternative secondallelic variants that include: (a) a first allele-specific primer; (b) asecond allele-specific primer; (c), a first locus-specific primer; (d) asecond locus-specific primer; (e) a first allele-specific blocker probe;(f) a second allele-specific blocker probe; and (g) a polymerase. Insome embodiments of the disclosed kits, the kit further comprises afirst locus-specific detector probe and a second locus-specific detectorprobe.

In another aspect, the present invention provides kits that include twoor more containers comprising the following components independentlydistributed in one of the two or more containers: (i) a firstallele-specific primer, wherein an allele-specific nucleotide portion ofthe first allele-specific primer is complementary to the first allelicvariant of a target sequence; and (ii) a first allele-specific blockerprobe that is complementary to a region of the target sequencecomprising the second allelic variant, wherein said region encompasses aposition corresponding to the binding position of the allele-specificnucleotide portion of the first allele-specific primer, and wherein thefirst allele-specific blocker probe comprises a minor groove binder.

In some illustrative embodiments, the kits can further include alocus-specific primer that is complementary to a region of the targetsequence that is 3′ from the first allelic variant and on the oppositestrand.

In other embodiments, the kits can further include a detector probe.

In some embodiments, the compositions, methods, and/or kits can be usedin detecting circulating cells in diagnosis. In one embodiment, thecompositions, methods, and/or kits can be used to detect tumor cells inblood for early cancer diagnosis. In some embodiments, the compositions,methods, and/or kits can be used for cancer or disease-associatedgenetic variation or somatic mutation detection and validation. In someembodiments, the compositions, methods, and/or kits can be used forgenotyping tera-, tri- and di-allelic SNPs. In other embodiments, thecompositions, methods, and/or kits can be used for identifying single ormultiple nucleotide insertion or deletion mutations. In someembodiments, the compositions, methods, and/or kits can be used for DNAtyping from mixed DNA samples for QC and human identification assays,cell line QC for cell contaminations, allelic gene expression analysis,virus typing/rare pathogen detection, mutation detection from pooledsamples, detection of circulating tumor cells in blood, and/or prenataldiagnostics.

In some embodiments, the compositions, methods, and/or kits arecompatible with various instruments such as, for example, SDSinstruments from Applied Biosystems (Foster City, Calif.).

Allele-Specific Primers

Allele-specific primers (ASPs) designed with low Tms exhibit increaseddiscrimination of allelic variants. In some embodiments, theallele-specific primers are short oligomers ranging from about 15-30,such as about 16-28, about 17-26, about 18-24, or about 20-22, or anyrange in between, nucleotides in length. In some embodiments, the Tm ofthe allele-specific primers range from about 50° C. to 70° C., such asabout 52° C. to 68° C., about 54° C. to 66° C., about 56° C. to 64° C.,about 58° C. to 62° C., or any temperature in between (e.g., 53° C., 54°C., 55° C., 56° C.). In other embodiments, the Tm of the allele-specificprimers is about 3° C. to 6° C. higher than the anneal/extendtemperature of the PCR cycling conditions employed during amplification.

Low allele-specific primer concentration can also improve selectivity.Reduction in concentration of allele-specific primers below 900 nM canincrease the delta Ct between matched and mismatched sequences. In someembodiments of the disclosed compositions, the concentration ofallele-specific primers ranges from about 20 nM to 900 nM, such as about50 nM to 700 nM, about 100 nM to 500 nM, about 200 nM to 300 nM, about400 nM to 500 nM, or any range in between. In some exemplaryembodiments, the concentration of the allele-specific primers is betweenabout 200 nM to 400 nM.

In some embodiments, allele-specific primers can comprise anallele-specific nucleotide portion that is specific to the target alleleof interest. The allele-specific nucleotide portion of anallele-specific primer is complementary to one allele of a gene, but notanother allele of the gene. In other words, the allele-specificnucleotide portion binds to one or more variable nucleotide positions ofa gene that is nucleotide positions that are known to include differentnucleotides for different allelic variants of a gene. Theallele-specific nucleotide portion is at least one nucleotide in length.In exemplary embodiments, the allele-specific nucleotide portion is onenucleotide in length. In some embodiments, the allele-specificnucleotide portion of an allele-specific primer is located at the 3′terminus of the allele-specific primer. In other embodiments, theallele-specific nucleotide portion is located about 1-2, 3-4, 5-6, 7-8,9-11, 12-15, or 16-20 nucleotides in from the 3′ most-end of theallele-specific primer.

Allele-specific primers designed to target discriminating bases can alsoimprove discrimination of allelic variants. In some embodiments, thenucleotide of the allele-specific nucleotide portion targets a highlydiscriminating base (e.g., for detection of A/A, A/G, G/A, G/G, A/C, orC/A alleles). Less discriminating bases, for example, may involvedetection of C/C, T/C, G/T, T/G, C/T alleles. In some embodiments, forexample when the allele to be detected involves A/G or C/T SNPs, A or Gmay be used as the 3′ allele-specific nucleotide portion of theallele-specific primer (e.g., if A or T is the major allele), or C or Tmay be used as the 3′ allele-specific nucleotide portion of theallele-specific primer (e.g., if C or G is the major allele). In otherembodiments, A may be used as the nucleotide-specific portion at the 3′end of the allele specific primer (e.g., the allele-specific nucleotideportion) when detecting and/or quantifying A/T SNPs. In otherembodiments, G may be used as the nucleotide-specific portion at the 3′end of the allele specific primer when detecting and/or quantifying C/GSNPs.

In some embodiments, the allele-specific primer can comprise atarget-specific portion that is specific to the polynucleotide sequence(or locus) of interest. In some embodiments the target-specific portionis about 75-85%, 85-95%, 95-99% or 100% complementary to the targetpolynucleotide sequence of interest. In some embodiments, thetarget-specific portion of the allele-specific primer can comprise theallele-specific nucleotide portion. In other embodiments, thetarget-specific portion is located 5′ to the allele-specific nucleotideportion. The target-specific portion can be about 4-30, about 5-25,about 6-20, about 7-15, or about 8-10 nucleotides in length. In someembodiments, the Tm of the target specific portion is about 5° C. belowthe anneal/extend temperature used for PCR cycling. In some embodiments,the Tm of the target specific portion of the allele-specific primerranges from about 51° C. to 60° C., about 52° C. to 59° C., about 53° C.to 58° C., about 54° C. to 57° C., about 55° C. to 56° C., or about 50°C. to about 60° C.

In some embodiments of the disclosed methods and kits, thetarget-specific portion of the first allele-specific primer and thetarget-specific portion of the second allele-specific primer comprisethe same sequence. In other embodiments, the target-specific portion ofthe first allele-specific primer and the target-specific portion of thesecond allele-specific primer are the same sequence.

In some embodiments, the allele-specific primer comprises a tail.Allele-specific primers comprising tails, enable the overall length ofthe primer to be reduced, thereby lowering the Tm without significantimpact on assay sensitivity.

In some exemplary embodiments, the tail is on the 5′ terminus of theallele-specific primer. In some embodiments, the tail is located 5′ ofthe target-specific portion and/or allele-specific nucleotide portion ofthe allele-specific primer. In some embodiments, the tail is about65-75%, about 75-85%, about 85-95%, about 95-99% or about 100%non-complementary to the target polynucleotide sequence of interest. Insome embodiments the tail can be about 2-40, such as about 4-30, about5-25, about 6-20, about 7-15, or about 8-10 nucleotides in length. Insome embodiments the tail is GC-rich. For example, in some embodimentsthe tail sequence is comprised of about 50-100%, about 60-100%, about70-100%, about 80-100%, about 90-100% or about 95-100% G and/or Cnucleotides.

The tail of the allele-specific primer may be configured in a number ofdifferent ways, including, but not limited to a configuration wherebythe tail region is available after primer extension to hybridize to acomplementary sequence (if present) in a primer extension product. Thus,for example, the tail of the allele-specific primer can hybridize to thecomplementary sequence in an extension product resulting from extensionof a locus-specific primer.

In some embodiments of the disclosed methods and kits, the tail of thefirst allele-specific primer and the tail of the second allele-specificprimer comprise the same sequence. In other embodiments, the 5′ tail ofthe first allele-specific primer and the 5′ tail of the secondallele-specific primer are the same sequence.

Allele-Specific Blocker Probes

Allele-specific blocker probes (or ASBs) (herein sometimes referred toas “blocker probes”) may be designed as short oligomers that aresingle-stranded and have a length of 100 nucleotides or less, morepreferably 50 nucleotides or less, still more preferably 30 nucleotidesor less and most preferably 20 nucleotides or less with a lower limitbeing approximately 5 nucleotides.

In some embodiments, the Tm of the blocker probes range from 58° C. to70° C., 61° C. to 69° C., 62° C. to 68° C., 63° C. to 67° C., 64° C. to66° C., or about 60° C. to about 63° C., or any range in between. In yetother embodiments, the Tm of the allele-specific blocker probes is about3° C. to 6° C. higher than the anneal/extend temperature in the PCRcycling conditions employed during amplification.

In some embodiments, the blocker probes are not cleaved during PCRamplification. In some embodiments, the blocker probes comprise anon-extendable blocker moiety at their 3′-ends. In some embodiments, theblocker probes can further comprise other moieties (including, but notlimited to additional non-extendable blocker moieties, quenchermoieties, fluorescent moieties, etc.) at their 3′-end, 5′-end, and/orany internal position in between. In some embodiments, the alleleposition is located about 5-15, such as about 5-11, about 6-10, about7-9, about 7-12, or about 9-11, such as about 6, about 7, about 8, about9, about 10, or about 11 nucleotides away from the non-extendableblocker moiety of the allele-specific blocker probes when hybridized totheir target sequences. In some embodiments, the non-extendable blockermoiety can be, but is not limited to, an amine (NH₂), biotin, PEG, DPI₃,or PO₄. In some preferred embodiments, the blocker moiety is a minorgroove binder (MGB) moiety. (The oligonucleotide-MGB conjugates of thepresent invention are hereinafter sometimes referred to as “MGB blockerprobes” or “MGB blockers.”)

As disclosed herein, the use of MGB moieties in allele-specific blockerprobes can increase the specificity of allele-specific PCR. Onepossibility for this effect is that, due to their strong affinity tohybridize and strongly bind to complementary sequences of single ordouble stranded nucleic acids, MGBs can lower the Tm of linkedoligonucleotides (See, for example, Kutyavin, I., et al., Nucleic AcidsRes., 2000, Vol. 28, No. 2: 655-661). Oligonucleotides comprising MGBmoieties have strict geometric requirements since the linker between theoligonucleotide and the MGB moiety must be flexible enough to allowpositioning of the MGB in the minor groove after DNA duplex formation.Thus, MGB blocker probes can provide larger Tm differences betweenmatched versus mismatched alleles as compared to conventional DNAblocker probes.

In general, MGB moieties are molecules that bind within the minor grooveof double stranded DNA. Although a generic chemical formula for allknown MGB compounds cannot be provided because such compounds havewidely varying chemical structures, compounds which are capable ofbinding in the minor groove of DNA, generally speaking, have a crescentshape three dimensional structure. Most MGB moieties have a strongpreference for A-T (adenine and thymine) rich regions of the B form ofdouble stranded DNA. Nevertheless, MGB compounds which would showpreference to C-G (cytosine and guanine) rich regions are alsotheoretically possible. Therefore, oligonucleotides comprising a radicalor moiety derived from minor groove binder molecules having preferencefor C-G regions are also within the scope of the present invention.

Some MGBs are capable of binding within the minor groove of doublestranded DNA with an association constant of 10³M⁻¹ or greater. Thistype of binding can be detected by well-established spectrophotometricmethods such as ultraviolet (UV) and nuclear magnetic resonance (NMR)spectroscopy and also by gel electrophoresis. Shifts in UV spectra uponbinding of a minor groove binder molecule and NMR spectroscopy utilizingthe “Nuclear Overhauser” (NOSEY) effect are particularly well known anduseful techniques for this purpose. Gel electrophoresis detects bindingof an MGB to double stranded DNA or fragment thereof, because upon suchbinding the mobility of the double stranded DNA changes.

A variety of suitable minor groove binders have been described in theliterature. See, for example, Kutyavin, et al. U.S. Pat. No. 5,801,155;Wemmer, D. E., and Dervan P. B., Current Opinion in Structural Biology,7:355-361 (1997); Walker, W. L., Kopka, J. L. and Goodsell, D. S.,Biopolymers, 44:323-334 (1997); Zimmer, C.& Wahnert, U. Prog. Biophys.Molec. Bio. 47:31-112 (1986) and Reddy, B. S. P., Dondhi, S. M., andLown, J. W., Pharmacol. Therap., 84:1-111 (1999). In one group ofembodiments, the MGB is selected from the group consisting of CC1065analogs, lexitropsins, distamycin, netropsin, berenil, duocarmycin,pentamidine, 4,6-diamino-2-phenylindole andpyrrolo[2,1-c][1,4]benzodiazepines. A preferred MGB in accordance withthe present disclosure is DPI₃ (see U.S. Pat. No. 6,727,356, thedisclosure of which is incorporated herein by reference in itsentirety).

Suitable methods for attaching MGBs through linkers to oligonucleotidesor probes and have been described in, for example, U.S. Pat. Nos.5,512,677; 5,419,966; 5,696,251; 5,585,481; 5,942,610; 5,736,626;5,801,155 and 6,727,356. (The disclosures of each of which areincorporated herein by reference in their entireties.) For example,MGB-oligonucleotide conjugates can be synthesized using automatedoligonucleotide synthesis methods from solid supports having cleavablelinkers. In other examples, MGB probes can be prepared from an MGBmodified solid support substantially in accordance with the procedure ofLukhtanov et al. Bioconjugate Chern., 7: 564-567 (1996). (The disclosureof which is also incorporated herein by reference in its entirety.)According to these methods, one or more MGB moieties can be attached atthe 5′-end, the 3′-end and/or at any internal portion of theoligonucleotide.

The location of an MGB moiety within an MGB-oligonucleotide conjugatecan affect the discriminatory properties of such a conjugate. Anunpaired region within a duplex will likely result in changes in theshape of the minor groove in the vicinity of the mismatched base(s).Since MGBs fit best within the minor groove of a perfectly-matched DNAduplex, mismatches resulting in shape changes in the minor groove wouldreduce binding strength of an MGB to a region containing a mismatch.Hence, the ability of an MGB to stabilize such a hybrid would bedecreased, thereby increasing the ability of an MGB-oligonucleotideconjugate to discriminate a mismatch from a perfectly-matched duplex. Onthe other hand, if a mismatch lies outside of the region complementaryto an MGB-oligonucleotide conjugate, discriminatory ability forunconjugated and MGB-conjugated oligonucleotides of equal length isexpected to be approximately the same. Since the ability of anoligonucleotide probe to discriminate single base pair mismatchesdepends on its length, shorter oligonucleotides are more effective indiscriminating mismatches. The first advantage of the use ofMGB-oligonucleotides conjugates in this context lies in the fact thatmuch shorter oligonucleotides compared to those used in the art (i.e.,20-mers or shorter), having greater discriminatory powers, can be used,due to the pronounced stabilizing effect of MGB conjugation.Consequently, larger delta Tms of allele-specific blocker probes canimprove AS-PCR assay specificity and selectivity.

Blocker probes having MGB at the 5′ termini may have additionaladvantages over other blocker probes having a blocker moiety (e.g., MGB,PO₄, NH₂, PEG, or biotin) only at the 3′ terminus. This is at leastbecause blocker probes having MGB at the 5′ terminus (in addition to ablocking moiety at the 3′-end that prevents extension) will not becleaved during PCR amplification. Thus, the probe concentration can bemaintained at a constant level throughout PCR, which may help maintainthe effectiveness of blocking non-specific priming, thereby increasingcast-PCR assay specificity and selectivity (FIG. 3).

In some embodiments, as depicted in FIG. 4A, the allele-specific primerand/or the allele-specific blocker probe can comprise one or moremodified nucleobases or nucleosidic bases different from the naturallyoccurring bases (i.e., adenine, cytosine, guanine, thymine and uracil).In some embodiments, the modified bases are still able to effectivelyhybridize to nucleic acid units that contain adenine, guanine, cytosine,uracil or thymine moieties. In some embodiments, the modified base(s)may increase the difference in the Tm between matched and mismatchedtarget sequences and/or decrease mismatch priming efficiency, therebyimproving not only assay specificity, bust also selectivity.

Modified bases are considered to be those that differ from thenaturally-occurring bases by addition or deletion of one or morefunctional groups, differences in the heterocyclic ring structure (i.e.,substitution of carbon for a heteroatom, or vice versa), and/orattachment of one or more linker arm structures to the base. In someembodiments, all tautomeric forms of naturally occurring bases, modifiedbases and base analogues may also be included in the oligonucleotideprimers and probes of the invention.

Some examples of modified base(s) may include, for example, the generalclass of base analogues 7-deazapurines and their derivatives andpyrazolopyrimidines and their derivatives (described in PCT WO 90/14353;and U.S. application Ser. No. 09/054,630, the disclosures of each ofwhich are incorporated herein by reference in their entireties).Examples of base analogues of this type include, for example, theguanine analogue 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (ppG),the adenine analogue 4-amino-1H-pyrazolo[3,4-d]pyrimidine (ppA), and thexanthine analogue 1H-pyrazolo[4,4-d]pyrimidin-4(5H)-6(7H)-dione (ppX).These base analogues, when present in an oligonucleotide of someembodiments of this invention, strengthen hybridization and can improvemismatch discrimination.

Additionally, in some embodiments, modified sugars or sugar analoguescan be present in one or more of the nucleotide subunits of anoligonucleotide conjugate in accordance with the invention. Sugarmodifications include, but are not limited to, attachment ofsubstituents to the 2′, 3′ and/or 4′ carbon atom of the sugar, differentepimeric forms of the sugar, differences in the α- or β-configuration ofthe glycosidic bond, and other anomeric changes. Sugar moieties include,but are not limited to, pentose, deoxypentose, hexose, deoxyhexose,ribose, deoxyribose, glucose, arabinose, pentofuranose, xylose, lyxose,and cyclopentyl.

Locked nucleic acid (LNA)-type modifications, for example, typicallyinvolve alterations to the pentose sugar of ribo- anddeoxyribonucleotides that constrains, or “locks,” the sugar in theN-type conformation seen in A-form DNA. In some embodiments, this lockcan be achieved via a 2′-O, 4′-C methylene linkage in1,2:5,6-di-O-isopropylene-α-D-allofuranose. In other embodiments, thisalteration then serves as the foundation for synthesizing lockednucleotide phosphoramidite monomers. (See, for example, Wengel J., Acc.Chem. Res., 32:301-310 (1998), U.S. Pat. No. 7,060,809; Obika, et al.,Tetrahedron Lett 39: 5401-5405 (1998); Singh, et al., Chem Commun4:455-456 (1998); Koshkin, et al., Tetrahedron 54: 3607-3630 (1998), thedisclosures of each of which are incorporated herein by reference intheir entireties.)

In some preferred embodiments, the modified bases include8-Aza-7-deaza-dA (ppA), 8-Aza-7-deaza-dG (ppG),2′-Deoxypseudoisocytidine (iso dC), 5-fluoro-2′-deoxyuridine (fdU),locked nucleic acid (LNA), or 2′-O,4′-C-ethylene bridged nucleic acid(ENA) bases. Other examples of modified bases that can be used in theinvention are depicted in FIG. 4B and described in U.S. Pat. No.7,517,978 (the disclosure of which is incorporated herein by referencein its entirety).

Many modified bases, including for example, LNA, ppA, ppG, 5-Fluoro-dU(fdU), are commercially available and can be used in oligonucleotidesynthesis methods well known in the art. In some embodiments, synthesisof modified primers and probes can be carried out using standardchemical means also well known in the art. For example, in certainembodiments, the modified moiety or base can be introduced by use of a(a) modified nucleoside as a DNA synthesis support, (b) modifiednucleoside as a phosphoramidite, (c) reagent during DNA synthesis (e.g.,benzylamine treatment of a convertible amidite when incorporated into aDNA sequence), or (d) by post-synthetic modification.

In some embodiments, the primers or probes are synthesized so that themodified bases are positioned at the 3′ end. In some embodiments, themodified base are located between, 1-6 nucleotides, e.g., 2, 3, 4 or 5nucleotides away from the 3′-end of the allele-specific primer orblocker probe. In some preferred embodiments, the primers or probes aresynthesized so that the modified bases are positioned at the 3′-most endof the allele-specific primer or blocker probe.

Modified internucleotide linkages can also be present in oligonucleotideconjugates of the invention. Such modified linkages include, but are notlimited to, peptide, phosphate, phosphodiester, phosphotriester,alkylphosphate, alkanephosphonate, thiophosphate, phosphorothioate,phosphorodithioate, methylphosphonate, phosphoramidate, substitutedphosphoramidate and the like. Several further modifications of bases,sugars and/or internucleotide linkages, that are compatible with theiruse in oligonucleotides serving as probes and/or primers, will beapparent to those of skill in the art.

In addition, in some embodiments, the nucleotide units which areincorporated into the oligonucleotides of the allele-specific primersand/or blocker probes of the present invention may have a cross-linkingfunction (an alkylating agent) covalently bound to one or more of thebases, through a linking arm.

In some embodiments of the methods and kits, the first allele-specificblocker probe binds to the same strand or sequence as the firstallele-specific primer, while the second allele-specific blocker probebinds to the opposite strand and/or complementary sequence as the firstallele-specific primer.

Detector Probes

In some embodiments, detector probe is designed as short oligomersranging from about 15-30 nucleotides, such as about 16, about 18, about22, about 24, about 30, or any number in between. In some embodiments,the Tm of the detector probe ranges from about 60° C. to 80° C., about61° C. to 69° C., about 62° C. to 68° C., about 63° C. to 67° C., orabout 64° C. to 66° C., or any range in between.

In some embodiments, the detector probe is a locus-specific detectorprobes (LST). In other embodiments the detector probe is a 5′ nucleaseprobe. In some exemplary embodiments, the detector probe can comprisesan MGB moiety, a reporter moiety (e.g., FAM™, TET™, JOE™, VIC™, or SYBR®Green), a quencher moiety (e.g., Black Hole Quencher™ or TAMRA™), and/ora passive reference (e.g., ROX™). In some exemplary embodiments, thedetector probe is designed according to the methods and principlesdescribed in U.S. Pat. No. 6,727,356 (the disclosure of which isincorporated herein by reference in its entirety). In some exemplaryembodiments, the detector probe is a TaqMan® probe (Applied Biosystems,Foster City). In exemplary embodiments, the locus-specific detectorprobe can be designed according to the principles and methods describedin U.S. Pat. No. 6,727,356 (the disclosure of which is incorporatedherein by reference in its entirety). For example, fluorogenic probescan be prepared with a quencher at the 3′ terminus of a single DNAstrand and a fluorophore at the 5′ terminus. In such an example, the5′-nuclease activity of a Taq DNA polymerase can cleave the DNA strand,thereby separating the fluorophore from the quencher and releasing thefluorescent signal. In some embodiments, the detector probes arehybridized to the template strands during primer extension step of PCRamplification (e.g., at 60-65° C.). In yet other embodiments, an MGB iscovalently attached to the quencher moiety of the locus-specificdetector probes (e.g., through a linker).

In some embodiments of the disclosed methods and kits, the first andsecond detector probes are the same and/or comprise the same sequence orare the same sequence.

Locus-Specific Primers

In some embodiments, locus-specific primer (LSP) is designed as a shortoligomer ranging from about 15-30 nucleotides, such as about 16, about18, about 22, about 24, about 30, or any number in between. In someembodiments, the Tm of the locus-specific primer ranges from about 60°C. to 70° C., about 61° C. to 69° C., about 62° C. to 68° C., about 63°C. to 67° C., or about 64° C. to 66° C., or any range in between.

In some other embodiments of the disclosed methods and kits, the firstlocus-specific detector probe and/or second locus-specific detectorprobes comprise the same sequence or are the same sequence.

Additional Components

Polymerase enzymes suitable for the practice of the present inventionare well known in the art and can be derived from a number of sources.Thermostable polymerases may be obtained, for example, from a variety ofthermophilic bacteria that are commercially available (for example, fromAmerican Type Culture Collection, Rockville, Md.) using methods that arewell-known to one of ordinary skill in the art (See, e.g., U.S. Pat. No.6,245,533). Bacterial cells may be grown according to standardmicrobiological techniques, using culture media and incubationconditions suitable for growing active cultures of the particularspecies that are well-known to one of ordinary skill in the art (See,e.g., Brock, T. D., and Freeze, H., J. Bacteriol. 98(1):289-297 (1969);Oshima, T., and Imahori, K, Int. J. Syst. Bacteriol. 24(1):102-112(1974)). Suitable for use as sources of thermostable polymerases are thethermophilic bacteria Thermus aquaticus, Thermus thermophilus,Thermococcus litoralis, Pyrococcus furiosus, Pyrococcus woosii and otherspecies of the Pyrococcus genus, Bacillus stearothermophilus, Sulfolobusacidocaldarius, Thermoplasma acidophilum, Thermus flavus, Thermus ruber,Thermus brockianus, Thermotoga neapolitana, Thermotoga maritima andother species of the Thermotoga genus, and Methanobacteriumthermoautotrophicum, and mutants of each of these species. Preferablethermostable polymerases can include, but are not limited to, Taq DNApolymerase, Tne DNA polymerase, Tma DNA polymerase, or mutants,derivatives or fragments thereof.

Various Sources and/or Preparation Methods of Nucleic Acids

Sources of nucleic acid samples in the disclosed compositions, methodsand/or kits include, but are not limited to, human cells such ascirculating blood, buccal epithelial cells, cultured cells and tumorcells. Also other mammalian tissue, blood and cultured cells aresuitable sources of template nucleic acids. In addition, viruses,bacteriophage, bacteria, fungi and other micro-organisms can be thesource of nucleic acid for analysis. The DNA may be genomic or it may becloned in plasmids, bacteriophage, bacterial artificial chromosomes(BACs), yeast artificial chromosomes (YACs) or other vectors. RNA may beisolated directly from the relevant cells or it may be produced by invitro priming from a suitable RNA promoter or by in vitro transcription.The present invention may be used for the detection of variation ingenomic DNA whether human, animal or other. It finds particular use inthe analysis of inherited or acquired diseases or disorders. Aparticular use is in the detection of inherited diseases.

In some embodiments, template sequence or nucleic acid sample can begDNA. In other embodiments, the template sequence or nucleic acid samplecan be cDNA. In yet other embodiments, as in the case of simultaneousanalysis of gene expression by RT-PCR, the template sequence or nucleicacid sample can be RNA. The DNA or RNA template sequence or nucleic acidsample can be extracted from any type of tissue including, for example,formalin-fixed paraffin-embedded tumor specimens.

Preamplification

In some embodiments, additional compositions, methods and kits areprovided for “boosting” cast-PCR amplification reactions for limitedquantity specimens having very low nucleic acid copy number. In someembodiments, said compositions, methods and kits involve a two-stepamplification process comprising a first “booster” or pre-amplificationmultiplex reaction (see, for example, U.S. Pat. Nos. 6,605,451 and7,087,414 and U.S. Published Application No. 2004/0175733, thedisclosures of which are herein incorporated by reference in theirentireties), followed by a second single-plex (i.e., cast-PCR)amplification reaction.

In some preferred embodiments, the first step involves a multiplexreaction which uses at least two complete sets of primers (e.g., oneforward allele-1-specific primer, one forward allele-2-specific primerand one reverse locus-specific primer), each set of which is suitable oroperative for amplifying a specific polynucleotide of interest. In otherembodiments, the resultant multiplex products acquired in the first stepare divided into optimized secondary single-plex cast-PCR amplificationreactions, each containing at least one primer set previously used inthe first multiplexing step and then PCR amplified using the cast-PCRmethods described herein.

In other preferred embodiments, the first multiplex reaction is acast-PCR amplification reaction (although other well-known amplificationmethods such as, but not limited to PCR, RT-PCR, NASBA, SDA, TMA, CRCA,Ligase Chain Reaction, etc. can be used). In certain embodiments, thefirst multiplex reaction comprises a plurality of allele-specificprimers, and locus-specific primers, each group of which is specific fora particular allele of interest and designed according to the cast-PCRmethods described herein. Unlike single-plex cast-PCR reactions thatgenerate a single amplified sequence, multiplex cast-PCR amplificationreactions, by virtue of utilizing a plurality of different primer sets,can permit the simultaneous amplification of a plurality of differentsequences of interest in a single reaction. Because a plurality ofdifferent sequences is amplified simultaneously in a single reaction,the multiplex amplifications can effectively increase the concentrationor quantity of a sample available for downstream cast-PCR assays. Thus,in some preferred embodiments, significantly more analyses or assays canbe performed with a pre-amplified cast-PCR sample than could have beenperformed with the original sample.

The number of different amplification primer pairs utilized in themultiplex amplification is not critical and can range from as few astwo, to as many as tens, hundreds, thousands, or even more. Thus,depending upon the particular conditions, the multiplex amplificationspermit the simultaneous amplification of from as few as two to as manyas tens, hundreds, thousands, or even more polynucleotide sequences ofinterest.

The number of amplification cycles performed with a multiplexamplification may depend upon, among other factors, the degree ofamplification desired. The degree of amplification desired, in turn, maydepend upon such factors as the amount of polynucleotide sample to beamplified or the number of alleles or mutations to be detected usingsubsequent cast-PCR assays.

In preferred embodiments, it may be desirable to keep the multiplexamplification from progressing beyond the exponential phase or thelinear phase. Indeed, in some embodiments, it may be desirable to carryout the multiplex amplification for a number of cycles suitable to keepthe reaction within the exponential or linear phase. Utilization of atruncated multiplex amplification round can result in a sample having aboosted product copy number of about 100-1000 fold increase.

In many embodiments, pre-amplification permits the ability to performcast-PCR assays or analyses that require more sample, or a higherconcentration of sample, than was originally available. For example,after a 10×, 100×, 1000×, 10,000×, and so on, multiplex amplification,subsequent cast-PCR single-plex assays can then be performed using,respectively, a 10×, 100×, 1000×, 10,000×, and so on, less samplevolume. In some embodiments, this allows each single-plex cast-PCRreaction to be optimized for maximum sensitivity and requires only onemethod of detection for each allele analyzed. This can be a significantbenefit to cast-PCR analysis since, in some embodiments, it allows forthe use of off-the-shelf commercially available cast-PCR reagents andkits to be pooled together and used in a multiplex amplificationreaction without extensive effort toward or constraints againstredesigning and/or re-optimizing cast-PCR assays for any given targetsequence. Moreover, in some embodiments, the ability to carry out amultiplex amplification with reagents and kits already optimized forcast-PCR analysis permits the creation of multiplex amplificationreactions that are ideally correlated or matched with subsequentsingle-plex cast-PCR assays.

The following examples are intended to illustrate but not limit theinvention.

EXAMPLES I. General Cast-PCR Assay Design

The general schema for the cast-PCR assays used in the followingexamples is illustrated in FIG. 1. For each SNP that was analyzed,allele-specific primers (ASPs) were designed to target a first allele(i.e. allele-1) and a second allele (i.e. allele-2). The cast-PCR assayreaction mixture for allele-1 analysis included a 5′-tailedallele-1-specific primer (ASP1), one MGB allele-2 blocker probe (MGB2),one common locus-specific TaqMan probe (LST) and one commonlocus-specific primer (LSP). The cast-PCR assay reaction mixture foranalysis of allele-2 included a 5′-tailed allele-2-specific primer(ASP2), one MGB allele-1 blocker probe (MGB1), one common locus-specificTaqMan probe (LST) and one common locus-specific primer (LSP).

II. Reaction Conditions

Each assay reaction mixture (10 μl total) contained 1× TaqMan GenotypingMaster Mixture (Applied Biosystems, Foster City, Calif.; P/N 437135),0.5 ng/μL genomic DNA or 1 million copies of plasmid DNA (or asindicated otherwise), 300 nM (unless specified otherwise) tailed-, or insome cases untailed-, allele-specific primer (ASP1 for detection ofallele-1 or ASP2 for detection of allele-2), 200 nM TaqMan probe (LST),900 nM locus-specific primer (LSP), 150 nM allele-specific MGB blockerprobe (MGB1 for detection of allele-2 or MGB2 for detection ofallele-1). The reactions were incubated in a 384-well plate at 95° C.for 10 minutes, then for 5 cycles at 95° C. for 15 seconds and 58° C.for 1 minute, then by 45 cycles at 95° C. for 15 seconds and 60° C. for1 minute. All reactions were run in duplicate or higher replication inan ABI PRISM 7900HT® Sequence Detection System, according to themanufacturer's instructions.

The 2-stage cycling protocol used in the following examples for cast-PCRamplification reactions is different from conventional allele-specificPCR (AS-PCR). The 2-stage cycling protocol comprises an initial 5 cyclesat a lower annealing/extension temperature (e.g., 58° C.), followed by45 standard cycles at a higher annealing/extension temperature (e.g.,60° C.). Due to the lower Tm of cast-PCR allele-specific primers (e.g.,53-56° C.), PCR is not optimal at standard annealing/extensionconditions (e.g., 60° C.). Consequently, lower annealing/extensiontemperatures used during the initial 5 cycles increases overall cast-PCRefficiency.

III. Nucleic Acid Samples

Plasmids containing specific SNP sequences were designed and orderedfrom BlueHeron (Bothell, Wash.). (See Table 1 for a list of plasmidscomprising SNPs used in some of the following examples.) The plasmidswere quantified using TaqMan RNase P Assay (Applied Biosystems, FosterCity, Calif.; P/N 4316838) according to the manufacturer's instructionsand were used as templates (See Table 1, RNase P Control) to validatesensitivity, linear dynamic range, specificity, and selectivity of thegiven assays.

Genomic DNAs were purchased from Coriell Institute for Medical Research(Camden, N.J.; NA17203, NA17129, NA17201). The genotypes of target SNPswere validated with TaqMan SNP Genotyping Assays (Applied Biosystems,Foster City, Calif.; P/N 4332856) according to the manufacturer'sinstructions.

IV. Modified Oligonucleotides

Modified bases were purchased from Berry and Associates (ppA: P/N BA0239; ppG: P/N BA 0242; fdU: P/N BA 0246; and iso dC: P/N BA 0236) orExicon (LNA-T Amidite: P/N EQ-0064; LNA-mC Amidite: P/N EQ-0066; LNA-GAmidite: P/N EQ-0082; and LNA-A Amidite: P/N EQ-0063). Oligonucleotidescomprising the modified nucleotides at their 3′ ends were synthesizedaccording to the manufacturer's instructions.

TABLE 1Plasmid SNP Sequences (target alleles are indicated in brackets). Table 1 disclosesSEQ ID NOS 1-27, respectively, in order of appearance. SNP ID SequenceCV11201742GCTCTGCTTCATTCCTGTCTGAAGAAGGGCAGATAGTTTGGCTGCTCCTGTG[C/T]TGTCACCTGCAATTCTCCCTTATCAGGGCCATTGGCCTCTCCCTTCTCTCTGTGAGGGATATTTTCTCTGACTTGTCAATCCACATCTTCCCV11349123GGCTTGCAATGGCTCCAACCGGAAGGGCGGTGCTCGAGCTGTGGTGCGTGC[C/T]GCTAAGTTGTGCGTTCCAGGGTGCACTCGC CV1207700GCAACTATACCCTTGATGGATGGAGATTTA[C/T]GCAATGTGTTTTACTGGGTAGAGTGACAGACCTTCV25594064CCTGAACTTATTTGGCAAGAGCGATGAGTACTCTTAAAATTACTATCTGGAAATTATATTATTTAGAATCTGCCAATTACCTAGATCCCCCCT[C/G]AACAATTGTTTCACCAAGGAACTTCCTGAA CV25639181GAATTGGTTGTCTCCTTATGGGAACTGGAAGTATTTTGACA[G/T]CTTTACCACATTTCTTCATGGGATAGTAAGTGTTAAACAGCTCTGAGCCATTTATTATCAGCTACTTGTAAATTAGCAGTAGAATTTTATTTTTATACTTGTAAGTGGGCAGTTACCTTTTGAGAGGAATACCTATAG RNaseP ControlGCGGAGGGAAGCTCATCAGTGGGGCCACGAGCTGAGTGCGTCCTGTCACTCCACTCCCATGTCCCTTGGGAAGGTCTGAGACTAGGG BRAF-1799TATACTACACCTCAGATATATTTCTTCATGAAGACCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAG[T/A]GAAATCTCGATGGAGTGGGTCCCATCAGTTTGAACAGTTGTCTGGATCCATTTTGTGGATGGTAAGAATTGAGGCTATTTTTCCACTGATTAAATTTTTGGCCCTGAGATGCTGCTGAGTT CTNNB1-121AGTGCTAATACTGTTTCGTATTTATAGCTGATTTGATGGAGTTGGACATGGCCATGGAACCAGACAGAAAAGCGGCTGTTAGTCACTGGCAGCAACAGTCTTACCTGGACTCTGGAATCCATTCTGGTGCCACT[A/G]CCACAGCTCCTTCTCTGAGTGGTAAAGGCAATCCTGAGGAAGAGGATGTGGATACCTCCCAAGTC CTNNB1-134CTTTTGATGGAGTTGGACATGGCCATGGAACCAGACAGAAAAGCGGCTGTTAGTCACTGGCAGCAACAGTCTTACCTGGACTCTGGAATCCATTCTGGTGCCACTACCACAGCTCCTT[C/T]TCTGAGTGGTAAAGGCAATCCTGAGGAAGAGGATGTGGATACCTCCCAAGTCCTGTATGAGTGGGAA EGFR-2369CTGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCA[C/T]GCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACAATATTGGCTCCCAGTACCTGCTCAACTGGTGTGTGCAGATCGCAAAGGTAATCAGGGAAGGGA EGFR-2573TGGCATGAACTACTTGGAGGACCGTCGCTTGGTGCACCGCGACCTGGCAGCCAGGAACGTACTGGTGAAAACACCGCAGCATGTCAAGATCACAGATTTTGGGC[T/G]GGCCAAACTGCTGGGTGCGGAAGAGAAAGAATACCATGCAGAAGGAGGCAAAGTAAGGAGGTG KRAS-176CGCAGGATTCCTACAGGAAGCAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGACACAG[C/G]AGGTCAAGAGGAGTACAGTGCAATGAGGGACCAGTACATGAGGACTGGGGAGGGCTTTCTTTGTGTATTTGCCATAAATAATACTAAATCATTTGAAGATATTC KRAS-183ACACAGGAAGCAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGACACAGCAGGTCA[A/C]GAGGAGTACAGTGCAATGAGGGACCAGTACATGAGGACTGGGGAGGGCTTTCTTTGTGTATTTGCCATAAATAATACTAAATCATTTGAAGATATTCACCATTATAGGTGGGTTTAAATTGAATATAATAAGCTGACATTAA KRAS-34GATATTAACCTTATGTGTGACATGTTCTAATATAGTCACATTTTCATTATTTTTATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCT[G/A]GTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGA KRAS-35GATATTAACCTTATGTGTGACATGTTCTAATATAGTCACATTTTCATTATTTTTATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTG[G/A]TGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATC KRAS-38GACATTATTTTTATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTG[G/A]CGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGGTGCAGGACCATTCTTTGATACAGATAAAGGTTTCTCTGACCATTTTCATGAGTACTTAT NRAS-181CAATTCTTACAGAAAACAAGTGGTTATAGATGGTGAAACCTGTTTGTTGGACATACTGGATACAGCTGGA[C/A]AAGAAGAGTACAGTGCCATGAGAGACCAATACATGAGGACAGGCGAAGGCTTCCTCTGTGTATTTGCCATCAATAATAGCAAGTCATTTGCGGATATTAACCTCTACAGGTACTAGGAGCATTATTTTCTCTGAAAGGATGNRAS-183ATTTACAGAAAACAAGTGGTTATAGATGGTGAAACCTGTTTGTTGGACATACTGGATACAGCTGGACA[A/T]GAAGAGTACAGTGCCATGAGAGACCAATACATGAGGACAGGCGAAGGCTTCCTCTGTGTATTTGCCATCAATAATAGCAAGTCATTTGCGGATATTAACCTCTACAGGTACTAGGAGCATTATTTTCTCTGAAAGGATG NRAS-35GATGGTTTCCAACAGGTTCTTGCTGGTGTGAAATGACTGAGTACAAACTGGTGGTGGTTGGAGCAG[G/A]TGGTGTTGGGAAAAGCGCACTGACAATCCAGCTAATCCAGAACCACTTTGTAGATGAATATGATCCCACCATAGAGGTGAGGCCCAGTGGTAGCCCG NRAS-38GATTTCCAACAGGTTCTTGCTGGTGTGAAATGACTGAGTACAAACTGGTGGTGGTTGGAGCAGGTG[G/A]TGTTGGGAAAAGCGCACTGACAATCCAGCTAATCCAGAACCACTTTGTAGATGAATATGATCCCACCATAGAGGTGAGGCCCAGTGGTAGCCC TP53-524GAGGCACCCGCGTCCGCGCCATGGCCATCTACAAGCAGTCACAGCACATGACGGAGGTTGTGAGGC[G/A]CTGCCCCCACCATGAGCGCTGCTCAGATAGCGATGGTGAGCAGCTGGGGCTGGAGAGACGACAGGGCTGGTTGCCCAGGGTCCCCAGGCCTCTGATTCCTCACTGATTGCTCTTAGGTCTGGCC TP53-637CTCCTCCTCAGCATCTTATCCGAGTGGAAGGAAATTTGCGTGTGGAGTATTTGGATGACAGAAACACTTTT[C/T]GACATAGTGTGGTGGTGCCCTATGAGCCGCCTGAGGTCTGGTTTGCAACTGGGGTCTCTGGGAGGAGGGGTTACTAGGGTGGTTGTCAGTGGCCCTC TP53-721TGCTTGGGCCTGTGTTATCTCCTAGGTTGGCTCTGACTGTACCACCATCCACTACAACTACATGTGTAACAGT[T/G]CCTGCATGGGCGGCATGAACCGGAGGCCCATCCTCACCATCATCACACTGGAAGACTCCAGGTCAGGAGCCATGCTTGCCACCCTGCACACTGGCCTGCTGTGCCCCAGCCTC TP53-733GATAGGTTGGCTCTGACTGTACCACCATCCACTACAACTACATGTGTAACAGTTCCTGCATGGGC[G/A]GCATGAACCGGAGGCCCATCCTCACCATCATCACACTGGAAGACTCCAGGTCAGGAGCCACTTGCCACCCTGCACACTGGAGCCTGCTGTGCCCCAGCCTC TP53-742CTCTGACTGTACCACCATCCACTACAACTACATGTGTAACAGTTCCTGCATGGGCGGCATGAAC[C/T]GGAGGCCCATCCTCACCATCATCACACTGGAAGACTCCAGGTCAGGAGCCACTTGCCACCCTGCACACTGGCCTGCTGTGCCTCCCAGCCTCTGCTTGCCTC TP53-743GATGACTGTACCACCATCCACTACAACTACATGTGTAACAGTTCCTGCATGGGCGGCATGAACC[G/A]GAGGCCCATCCTCACCATCATCACACTGGAAGACTCCAGGTCAGGAGCCACTTGCCACCCTGCACACTGGCCTGCTGTGCCGACCAGCCTCTGCTTGCCTC TP53-817CTCCTCTTGCTTCTCTTTTCCTATCCTGAGTAGTGGTAATCTACTGGGACGGAACAGCTTTGAGGTG[C/T]GTGTTTGTGCCTGTCCTGGGAGAGACCGGCGCACAGAGGAAGAGAATCTCCGCAAGAAAGGGGAGCCTCACCACGAGCCTTGCCCCCAGGGAGCACTAAGCGAGGTAAGCAAData Analysis:

An automatic baseline and manual threshold of 0.2 were used to calculatethe threshold cycle (C_(t)) which is defined as the fractional cyclenumber at which the fluorescence passes the fixed threshold. PCRreactions were run for a total of 50 cycles. For cast-PCR reactions,there was a pre-run of five cycles at a lower annealing/extensiontemperature followed by an additional 45 cycles at a higherannealing/extension temperature. The ΔCt between amplification reactionsfor matched vs. mismatched sequences is defined as the specificity ofcast-PCR (ΔCt=C_(mismatch)−Ct_(match)). The larger the ΔCt betweenmismatched and matched targets, the better assay specificity. The2^(ΔCt) value was used to estimate the power of discrimination (orselectivity) which is equal to ½^(ΔCt) or, in some cases, calculated as% (½^(ΔCt)×100).

Example 1 Tailed Primers Improve Discrimination of Allelic Variants

The following example demonstrates that the application ofallele-specific primers comprising tails significantly improves thediscrimination of allelic variants.

In conventional AS-PCR, the discrimination of 3′ nucleotide mismatchesis largely dependent on the sequence surrounding the SNP and the natureof the allele. The ΔCt between the amplification reactions for matchedand mismatched primers varies. To improve the discrimination between theamplification of matched and mismatched sequences, allele-specificprimers were designed to comprise tails at their 5′ termini and thentested for their suitability in AS-PCR assays.

Assays were performed using the general experimental design and reactionconditions indicated above (with the exception that no blocker probeswere included and either tailed or non-tailed allele-specific primerswere added), using 0.5 ng/uL genomic DNA containing the hsv11711720 SNPcomprising one of three alleles (A, C, or T) as the nucleic acidtemplate (see Table 2A). The three genotypes are indicated in Table 2B.Primers and probes were designed according to the sequences shown inTable 3.

TABLE 2A Genomic DNA Sequence for hsv11711720 SNP(SEQ ID NO. 28) (target alleles are indicated in brackets).AGAAAATAACTAAGGGAAGGAGGAAAGTGGGGAGGAAGGAAGAACAGTGTGAAGACAATGGCCTGAAAACTGAAAAAGTCTGTTAAAGTTAATTATCAGTTTTTGAGTCCAAGAACTGGCTTTGCTACTTTCTGTAAGTTTCTAATTTACTGAATAAGCATGAAAAAGATTGCTTTGAGGAATGGTTATAAACACATTCTTAGAGCATAGTAAGCAGTAGGGAGTAACAAAATAACACTGATTAGAATACTTTACTCTACTTAATTAATCAATCATATTTAGTTTGACTCACCTTCCCAG[A/C/T]ACCTTCTAGTTCTTTCTTATCTTTCAGTGCTTGTCCAGACAACATTTTCATTTCAACAACTCCTGCTATTGCAATGATGGGTACAATTGCTAAGAGTAACAGTGTTAGTTGCCAACCATAGATGAAGGATATAATTATTCCTGTCCCAAGATTTGCTATATTCTGGGTAATTACAGCAAGCCTGGAACCTATAGCCTGCAAAACAAAACAAATTAGAGAAATTTTAAAAATATTATCTTCACAACTCATGCTTCTATTTTCTGAAAACTCACCTTCATGAG ACTATATTCATTATTTTAT

TABLE 2B Genotypes of Genomic DNA Sequence for hsv11711720 SNP GenomicDNA ID Genotype NA17203 AA NA17129 CC NA17201 TT

TABLE 3 List and Sequences of Primers and Probes(SEQ ID NOS 29-36, respectively, in order ofappearance) for genomic DNA: conventionalallele-specific primers (“ASP”); tailed allele-specific primers (“tailASP”); locus-specificTaqMan probe (LST); locus-specific primer (LSP).The nucleotides shown in lower case are thetailed portion of the primers. The nucleotide-specific portion of each allele-specific primeris at the 3′-most terminus of each primer (indicated in bold). TmPrimer/Probe ID Sequence (5′ to 3′) (° C.) 17129-ASPATATTTAGTTTGACTCACCTTCCCAGC 63.2 17129-tailASP accACTCACCTTTCCCAGC 63.017203-ASP ATATTTAGTTTGACTCACCTTCCCAGA 62.0 17203-tailASPaccACTCACCTTTCCCAGA 63.7 17201-ASP ATATTTAGTTTGACTCACCTTCCCAGT 62.217201-tailASP accACTCACCTTTCCCAGT 64.0 LST (6-FAM)-TGGACAAGCACTGAAAGA-67.4 (MGB) LSP GCAGGAGTTGTTGAAATGAAAATGTTG 62.5

As shown in Table 4, when using non-tailed ASPs (“ASP −tail”), thediscrimination of 3′ nucleotide mismatch is largely dependent on thenature of the allele, as a considerable range of ΔCt values is observeddepending on the identity of the 3′-terminal base. The range of ΔCtvalues between matched and mismatched nucleotides (“NT”) were from −0.1to 10. However, with tailed ASPs (ASP +tail), the discrimination of 3′nucleotide mismatch was significantly improved. In fact, as Table 4shows, the ΔCt value between matched and mismatched nucleotides wasconsistently equal to or greater than 10 when tailed ASPs were used. TheCt values for amplification of matched sequences using tailed ASPs werecomparable to those using conventional or non-tailed ASPs. These resultsindicate that tailed ASP, can improve the specificity of AS-PCR, but maynot improve the sensitivity of detection.

TABLE 4 Tailed allele-specific primers (“ASP”) significantly improvediscrimination of allelic variants. The specificity (“fold difference”)was calculated based on the difference between Ct values using tailedvs. untailed primers (2^((ΔCt(ASP+tail)−(ΔCt(ASP−tail))). The mismatchednucleotides of the 3′ allele-specific nucleotide portion of the ASPs(+/−tail) and the target allele are also indicated (“NT mismatch”).Specificity Improvement NT mismatch ΔCt (ASP − tail) ΔCt (ASP + tail)fold difference C-A 0.9 11.5 1552.1 C-T 1.2 11.5 1278.3 A-C 10.0 11.93.7 A-G 9.8 11.9 4.3 T-G 2.3 11.5 588.1 T-C −0.1 11.5 3104.2 Average 4.011.6 1088.5

Example 2 Low Primer Concentrations Improve Discrimination of AllelicVariants

Assays were performed using the general experimental design and reactionconditions indicated above, in the presence of 1 million copies ofplasmid DNA containing various SNP target sequences (see Table 1) and200 nM or 800 nM tailed ASP (as indicated). Assay primers and probeswere designed according to the sequences shown in FIG. 11A.

The effect of tailed ASP concentration on discrimination of allelicvariants is summarized in Table 5. The ΔCt between the amplificationreactions for matched and mismatched primers demonstrate that lowertailed ASP concentrations improve discrimination of allelic variants.

TABLE 5 Assay Results Using Different Concentrations of TailedAllele-specific Primers ΔCt ΔCt Specificity Plasmid (ASP @ (ASP @Improvement SNP ID 800 nM) 200 nM) (fold difference) CV11201742 14.115.2 2.14 CV11349123 8.2 10 3.48 CV1207700 5.2 6.6 2.64 CV25594064 20.119.1 0.5 CV25639181 11.9 12.9 2 Average 12.6 13.44 2.14

Example 3 Primers Designed with Reduced Tms Improves Discrimination ofAllelic Variants

Assays were performed using the general experimental design and reactionconditions indicated above, in the presence of 1 million copies ofplasmid DNA containing various SNP target sequences (see Table 1) usingtailed ASP with a higher Tm (˜57° C.) or tailed ASP with a lower Tm(˜53° C.). Assay primers and probes were designed according to thesequences shown in FIG. 11B-E (see FIG. 11B for higher Tm ASP and FIG.11C or lower Tm ASP).

The effect of allele-specific primer Tm on discrimination of allelicvariants is summarized in Table 6. The ΔCt of allele-specific primerswith a lower Tm are significantly higher than those of allele-specificprimers with a higher Tm. Allele-specific primers designed with reducedTms improved discrimination of allelic variants by as much as 118-foldin some cases or an average of about 13-fold difference.

TABLE 6 ΔCt Values Using Tailed ASPs with Lower Tm (~53° C.) or withHigher Tm (~57° C.) Specificity Plasmid ΔCt (ASP w/ ΔCt (ASP w/Improvement SNP ID Tm ~57° C.) Tm ~53° C.) (fold difference BRAF-1799TA12.2 19.1 118.9 CTNNB1-121AG 11.6 14.9 10.0 KRAS-176CG 18.8 22.5 13.1RAS-35GA 13.0 14.0 1 TP53-721TG 14.7 19.1 20.6 CTNNB1-134CT 8.6 14.144.8 EGFR-2369CT 9.7 10.7 2 KRAS-183AC 22.2 23.1 1.8 RAS-38GA 14.0 14.31.2 TP53-733GA 13.6 13.5 1.0 EGFR-2573TG 16.7 20.2 10.9 KRAS-34GA 1414.8 1.8 KRAS-38GA 11.2 14.4 8.9 RAS-181CA 24.0 27.1 8.6 TP53-742CT 9.18.0 0.5 KRAS-35GA 11.5 15.1 12.3 RAS-183AT 23.6 22.7 0.5 TP53-524GA 11.413.5 4.6 TP53-637CT 11.4 14.4 7.8 TP53-743GA 10.1 13.2 8.4 TP53-817CT13.6 13.9 1.2 Average 14.1 16.3 13.3

Example 4 Use of Blocker Probes Improves Discrimination of AllelicVariants

The following example illustrates that the use of MGB blocker probesimproves the discrimination between 3′ nucleotide mismatched and matchedprimers to target sequences in AS-PCR reactions.

Assays were performed using the general cast-PCR schema and reactionconditions indicated above, using 1 million copies of plasmid DNAcontaining various SNP target sequences (see Table 1) in the presence ofMGB blocker probes or in the absence of MGB blocker probes. Assayprimers and probes were designed according to the sequences shown inFIG. 11C-E.

To improve the selectivity of AS-PCR, blocker probes were synthesized tocomprise an MGB group at their 3′ terminus. (See, for example, Kutyavin,I. V., et al., Nucleic Acids Research, 2000, Vol. 28, No. 2: 655-661,U.S. Pat. Nos. 5,512,677; 5,419,966; 5,696,251; 5,585,481; 5,942,610 and5,736,626.)

The results of cast-PCR using MGB blocker probes are summarized in Table7. The ΔCt between cast-PCR with MGB blocker probes is larger than thatwithout MGB blocker probes. As shown, MGB blocker probes improve thediscrimination of allelic variants.

TABLE 7 MGB Blocker Probes Improve Discrimination of Allelic VariantsSpecificity ΔCt (no MGB ΔCt (+MGB Improvement SNP ID blocker) blocker)fold difference) BRAF-1799TA 11.4 14.9 11.5 CTNNB1-121AG 11.6 14.1 5.4KRAS-176CG 17.8 20.9 9 NRAS-35GA 13.9 14.3 1.4 TP53-721TG 12.5 14.7 4.4CTNNB1-134CT 6.7 10.2 11.6 EGFR-2369CT 7.7 10.1 5.3 KRAS-183AC 22.4 231.5 NRAS-38GA 14.5 14.6 1.1 TP53-733GA 13.2 14.4 2.3 EGFR-2573TG 18.221.8 11.6 KRAS-34GA 14.4 15.1 1.7 KRAS-38GA 11.9 15.1 1.7 NRAS-181CA19.3 24.2 30.2 TP53-742CT 12.7 13.6 1.9 KRAS-35GA 11.0 13.7 6.5NRAS-183AT 20.2 21.7 2.9 TP53-524GA 13.5 13.5 1 TP53-637CT 9.3 12.1 7.0TP53-743GA 9.9 11.5 3.1 TP53-817CT 12.6 13.2 1.5 Average 13.6 15.5 6.0

Example 5 Primers Designed to Target Discriminating Bases ImprovesDiscrimination of Allelic Variants

Assays were performed using the general cast-PCR schema and reactionconditions indicated above, in the presence of 1 million copies ofplasmid DNA containing SNP target sequences (see Table 1). Assay primersand probes were designed according to the sequences shown in FIG. 11C-E.

According to the data summarized in Table 8, the discrimination ofcast-PCR was dependent on the nature of the allele being analyzed. AsTable 8 indicates, the ΔCt between mismatched and matched sequences forallele-1 were different from ΔCt between mismatched and matchedsequences for allele-2. However, both A and G bases, as compared to a Tbase, were highly discriminating for allele-1 and allele-2 in all fourSNPs examined.

TABLE 8 Primers Designed to Target Discriminating Bases ImproveDiscrimination of Allelic Variants ASP design SNP allele-1 SNP allele-23′ NT 3′ NT ΔCt Specificity ΔCt Specificity of of Allele (Ct_mismatch −(fold Allele (Ct_mismatch − (fold SNP ID ASP1 ASP2 NT Ct_match)difference) NT Ct_match) difference) KRAS-38GA G A C 13.4 10809 T 8.2294 NRAS-181CA C A G 27.5 189812531 T 9.8 891 NRAS-183AT A T T 17.9244589 A 23.4 11068835 TP53-742CT C T G 12.3 5043 A 8.3 315

Example 6 Determination of the Sensitivity and Dynamic Range forCast-PCR

In this example, the sensitivity and dynamic range of cast-PCR wasdetermined by performing cast-PCR using various copy numbers of a targetplasmid.

Assays were performed using the general cast-PCR schema and reactionconditions indicated above, using 1×10⁰ (1 copy) to 1×10⁷ copies ofplasmid DNA containing the NRAS-181CA SNP target sequence (see Table 1).Assay primers and probes were designed according to the sequences shownin FIG. 11C-E.

As shown in FIG. 5, the use of tailed primers and MGB-blocker probesdoes not adversely affect the sensitivity of cast-PCR, as thesensitivity of cast-PCR is comparable to TaqMan assays which do notutilize tailed primers or blocker probes. Furthermore, FIG. 5 shows thatthe cast-PCR assay shows a linear dynamic range over at least 7 logs.

Example 7 Determination of the Specificity of Cast-PCR

In this example, the specificity of cast-PCR was determined by comparingthe amplification of particular alleles of KRAS using either matched ormismatched ASPs to a given allele in the presence of their correspondingblocker probes.

Assays were performed using the general cast-PCR schema and reactionconditions indicated above, using 1×10⁶ copies of plasmid DNA containingeither one of two alleles of the KRAS-183AC SNP target sequence (seeTable 1). Assay primers and probes were designed according to thesequences shown in FIG. 11C-E.

The left panel of FIG. 7 shows the an amplification plot of cast-PCR onallele-1 DNA using matched (A1) primers in the presence of A2 blockerprobes or mismatched (A2) primers in the presence of A1 blocker probes.The right hand panel shows a similar experiment in which cast-PCR wasperformed on allele-2 DNA. As indicated in the data summary in FIG. 7, arobust ΔCt values of over 20 were observed for cast-PCR on both allelesof KRAS-183AC tested. This corresponds to a specificity as determined bya calculation of 2^(ΔCt) of 9×10⁶, and 2×10⁶, respectively, for allele-1and allele-2. Furthermore, a calculation of selectivity (½^(ΔCt))indicates that values of 1/1.1×10⁷ and 1/5.0×10⁷ are observed forallele-1 and allele-2, respectively.

Example 8 Cast-PCR is Able to Detect a Single Copy Mutant DNA in OneMillion Copies of Wild Type DNA

In this example, the selectivity of cast-PCR, i.e., the ability ofcast-PCR to detect a rare mutant DNA in an excess of wild type DNA, wasdetermined.

Assays were performed using the general cast-PCR schema and reactionconditions indicated above, using various copy numbers of mutantKRAS-183AC plasmid DNA (1 copy to 1×10⁶ copies) mixed with 1×10⁶ copiesof wild type KRAS-183AC plasmid DNA (see Table 1). Assay primers andprobes were designed according to the sequences shown in FIG. 11C-E, andcast-PCR reactions were performed using wild type or mutantallele-specific primers and the corresponding MGB blocker probes.

FIG. 8 shows that cast-PCR is able to detect as little as one copy of amutant DNA sequence, even when surrounded by a million-fold excess of awild type sequence.

Example 9 Selectivity of Cast-PCR in Discriminating Tumor Cell DNA fromNormal Cell DNA

In this example, the selectivity of cast-PCR was determined byperforming assays on samples in which various amounts of tumor cellgenomic DNA were mixed with or “spiked” into genomic DNA from normalcells. DNA samples were extracted using QIAmp DNA Mini Prep Kits(Qiagen). Wild type DNA was extracted from the SW48 cell line and mutantDNA was extracted the H1573 cell line.

The mutant DNA contained the KRAS-G12A mutation (See FIG. 6). Thepercentage of tumor cell DNA in the spiked samples varied from 0.5 to100%. cast-PCR was used to detect the presence of tumor cell DNA whenpresent in these percentages.

Assays were performed using the general cast-PCR schema and reactionconditions indicated above, using 30 ng of gDNA per reaction. Assayprimers and probes were designed according to the sequencescorresponding to KRAS-G12A SNP ID, as shown in FIGS. 18A and 18B.

As shown in FIG. 9, tumor cell DNA, even when present only at a level of0.5% as compared to normal cell DNA, is easily detected using cast-PCR.

Example 10 Use of Cast-PCR to Detect Tumor Cells in Tumor Samples

In this example, cast-PCR was used to detect and determine thepercentage of tumor cells in tumor samples. Various normal and tumorsamples were obtained and assayed by cast-PCR for the presence of anumber of SNPs associated with cancer as shown in FIG. 10.

Assays were performed using the general cast-PCR schema and reactionconditions indicated above, using 5 ng of gDNA or 1.5 ng cDNA derivedfrom either normal or tumor samples. Assay primers and probescorresponding to the SNPs shown in FIG. 10 were designed according tothe sequences as shown in FIG. 11C-E.

The results shown in FIG. 10 indicate that cast-PCR has a low falsepositive rate as indicated by the failure of cast-PCR to detect thepresence of mutant cells in normal samples. In contrast, cast-PCR wasable to provide a determination of the percentage of tumor cells invarious tumor samples that ranged from just under 2% for a tumor samplecontaining the NRAS-183AT SNP to greater than 80% for a samplecontaining the CTNNB1-134CT SNP.

Example 11 Use of Pre-Amplification in Cast-PCR

The following example demonstrates that pre-amplification combined withcast-PCR methods enables detection of multiple alleles from a limitedamount of genomic DNA template.

Prior to conducting cast-PCR amplification, multiplex reactions wereperformed for 7 different KRAS mutations (see Table 9).

10 μl multiplex reactions were prepared in a single tube by combininginto one reaction 45 nM of each allele-specific primer (including theallele-1-specific primer and the allele-2-specific primer) and 45 nM ofeach locus-specific primer for each of the seven different KRAS SNPs, aslisted in FIGS. 18A and 18B, 0.1 ng/μL genomic DNA, and 1× Preamp MasterMix (Applied Biosystems, Foster City, Calif.; P/N 437135). The 10 μlpre-amplification reactions were then incubated in an Applied Biosystems9700 Thermocyler in a 96- or 384-well plate for 95° C. for 10 minutes,followed by 10 cycles of 95° C. for 15 seconds, 60° C. for 4 minutes,and 99.9° C. for 10 minutes, and then held at 4° C. Next, 190 μl of0.1×TE pH 8.0 was added to each 10 μl pre-amplification reaction (20×dilution). The diluted pre-amplification reaction products were thendirectly used in subsequent cast-PCR reactions or stored at −20° C. forat least one week prior to use.

TABLE 9KRAS SNP Sequences (SEQ ID NOS 37-42, respectively, in order of appearance)(target alleles are indicated in brackets). SNP ID SNP SequenceKRAS-G12A_GCTGACTGAATATAAACTTGTGGTAGTTGGAGCTG[G/C]TGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGGTGCAGGACCATTCTTTGATACA KRAS-G12R_GCTGACTGAATATAAACTTGTGGTAGTTGGAGCT[G/C]GTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGGTGCAGGACCATTCTTTGATACA KRAS-G12D_GATGACTGAATATAAACTTGTGGTAGTTGGAGCTG[G/A]TGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGGTGCAGGACCATTCTTTGATACA KRAS-G12S_GATGACTGAATATAAACTTGTGGTAGTTGGAGCT[G/A]GTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGGTGCAGGACCATTCTTTGATACA KRAS-G13D_GATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTG[G/A]CGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGGTGCAGGACCATTCTTTGATACA KRAS-G12C_GTGTGAGTTTGTATTAAAAGGTACTGGTGGAGTATNNGATAGTGTATTAACCTTATGTGTGACATGTTCTAATATAGTCACATTTTCATTATTTTTATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCT[G/T]GTGGCGTAGGCAAGAGT

Following pre-amplification, the diluted pre-amplification products werealiquotted into single-plex cast-PCR reactions. Individual assays wereperformed for each of the 7 different KRAS mutations using the generalexperimental design and reaction conditions indicated above (see sectionII of Examples). 10 μL cast-PCR reactions were run for assayscontaining, as the nucleic acid template, either 1 μL 20× dilutedpre-amplification reaction product (as prepared above) or, as acomparison, 0.07 ng genomic DNA. All assay primers and probes weredesigned according to the sequences shown in FIGS. 18A and 18B.

As shown in FIG. 12, for assays without pre-amplification the averageΔCt of the 7 tested KRAS mutations was 12.0, whereas for assays usingpre-amplification the average ΔCt was 17.0. Thus, the ΔΔCt between thetwo gave about a 5 fold improvement for the pre-amplified reactions overthose without pre-amplification.

In an ideal situation (where PCR efficiency=100%), the copy number ofthe target gene increases about 1000 fold in 10 cycles. Under theseconditions, if the starting copy number in a pre-amplification reactionis 0.1 ng/μL (or approximately 33 copies/μL), then after 10 cycles thecopy number increases to approximately 33,000 copies/μL. In the exampleabove, the copy number of a 20 fold diluted pre-amplification product isestimated to be approximately 1,650 copies/μL. Therefore, after adding 1μL of 1,650 copies/μL diluted pre-amplification product into a cast-PCRreaction (final volume of 10 μL), the concentration of the cast-PCRproducts is approximately 165 copies/μL. Based on this estimation, 10 μLof pre-amplification products from 1 ng/μL genomic DNA can be diluted byas much as 200 fold, and still provide up to 2000 μL of nucleic acidtemplate for use in subsequent cast-PCR reactions.

Example 12 Effect of Tailed ASP on Cast-PCR Specificity

Assays were performed using the general cast-PCR schema and reactionconditions indicated above (see section II of Examples), using 1 millioncopies of plasmid DNA containing various SNP target sequences (see Table10). Assay primers and probes were designed according to the sequencesshown in FIG. 19A-D. For each SNP analyzed, the blocker probes,locus-specific probes and locus-specific primers were the same and onlythe allele-specific primers varied (e.g., tailed or non-tailed).

TABLE 10Plasmid SNP Sequences (SEQ ID NOS 43-78, respectively, in order of appearance)(target alleles are indicated in brackets). SNP ID SequenceBRAF-1799TA_TPATATTTCTTCATGAAGACCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAG[T/A]GAAATCTCGATGGAGTGGGTCCCATCAGTTTGAACAGTTGTCTGGATCCATTTTG CTNNB1-121AG_TPGTATCCACATCCTCTTCCTCAGGATTGCCTTTACCACTCAGAGAAGGAGCTGTGG[A/G]AGTGGCACCAGAATGGATTNCAGAGTNCAGGTAAGACTGTTGCTGCCAGTGACTA CTNNB1-134CT_TPCAGGACTTGGGAGGTATCCACATCCTCTTCCTCAGGATTGCCTTTACCACTCAGA[C/T]AAGGAGCTGTGGTAGTGGCACCAGAATGGATTNCAGAGTNCAGGTAAGACTGTTG EGFR-2369CT_TPCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCA[C/T]GCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACAATA EGFR-2573 TG_TPTTACTTTGCCTCCTTCTGCATGGTATTCTTTCTCTTCCGCACCCAGCAGTTTGGCC[T/G]GCCCAAAATCTGTGATCTTGACATGCTGCGGTGTTTTCACCAGTACGTTCCTGG KRAS-176CG_TPAGGAAGCAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGACACAG[C/G]AGGTCANGAGGAGTACAGTGCAATGAGGGACCAGTACATGAGGACTGGGGAGGGC KRAS-183AC_TPAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGACACAGCAGGTCA[A/C]GAGGAGTACAGTGCAATGAGGGACCAGTACATGAGGACTGGGGAGGGCTTTCTTT KRAS-34GA_TPCCACAAAATGATTCTGAATTAGCTGTATCGTCAAGGCACTCTTGCCTACGCCAC[G/A]AGCTCCAACTACCACAAGTTTATATTCAGTCATTTTCAGCAGGCCTTATAATAAAA KRAS-35GA_TPTCCACAAAATGATTCTGAATTAGCTGTATCGTCAAGGCACTCTTGCCTACGCCA[G/A]CAGCTCCAACTACCACAAGTTTATATTCAGTCATTTTCAGCAGGCCTTATAATAAA KRAS-3GA_TPATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTG[G/A]CGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGA NRAS-181CA_TPAACAAGTGGTTATAGATGGTGAAACCTGTTTGTTGGACATACTGGATACAGCTGGA[C/A]AAGAAGAGTACAGTGCCATGAGAGACCAATACATGAGGACAGGCGAAGGCTTCC NRAS-183AT_TPCAAGTGGTTATAGATGGTGAAACCTGTTTGTTGGACATACTGGATACAGCTGGACA[A/T]GAAGAGTACAGTGCCATGAGAGACCAATACATGAGGACAGGCGAAGGCTTCCTC BRAF-1799TA_TPATATTTCTTCATGAAGACCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAG[T/A]GAAATCTCGATGGAGTGGGTCCCATCAGTTTGAACAGTTGTCTGGATCCATTTTG CTNNB1-121AG_TPGTATCCACATCCTCTTCCTCAGGATTGCCTTTACCACTCAGAGAAGGAGCTGTGG[A/G]AGTGGCACCAGAATGGATTNCAGAGTNCAGGTAAGACTGTTGCTGCCAGTGACTA CTNNB1-134CT_TPCAGGACTTGGGAGGTATCCACATCCTCTTCCTCAGGATTGCCTTTACCACTCAGA[C/T]AAGGAGCTGTGGTAGTGGCACCAGAATGGATTNCAGAGTNCAGGTAAGACTGTTG EGFR-2369CT_TPCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCA[C/T]GCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACAATA EGFR-2573 TG_TPTTACTTTGCCTCCTTCTGCATGGTATTCTTTCTCTTCCGCACCCAGCAGTTTGGCC[T/G]GCCCAAAATCTGTGATCTTGACATGCTGCGGTGTTTTCACCAGTACGTTCCTGG KRAS-176CG_TPAGGAAGCAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGACACAG[C/G]AGGTCANGAGGAGTACAGTGCAATGAGGGACCAGTACATGAGGACTGGGGAGGGC KRAS-183AC_TPAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGACACAGCAGGTCA[C/G]GAGGAGTACAGTGCAATGAGGGACCAGTACATGAGGACTGGGGAGGGCTTTCTTT KRAS-34GA_TPCCACAAAATGATTCTGAATTAGCTGTATCGTCAAGGCACTCTTGCCTACGCCAC[G/A]AGCTCCAACTACCACAAGTTTATATTCAGTCATTTTCAGCAGGCCTTATAATAAAA KRAS-35GA_TPTCCACAAAATGATTCTGAATTAGCTGTATCGTCAAGGCACTCTTGCCTACGCCA[G/A]CAGCTCCAACTACCACAAGTTTATATTCAGTCATTTTCAGCAGGCCTTATAATAAA KRAS-3GA_TPATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTG[G/A]CGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGA NRAS-181CA_TPAACAAGTGGTTATAGATGGTGAAACCTGTTTGTTGGACATACTGGATACAGCTGGA[C/A]AAGAAGAGTACAGTGCCATGAGAGACCAATACATGAGGACAGGCGAAGGCTTCC NRAS-183AT_TPCAAGTGGTTATAGATGGTGAAACCTGTTTGTTGGACATACTGGATACAGCTGGACA[A/T]GAAGAGTACAGTGCCATGAGAGACCAATACATGAGGACAGGCGAAGGCTTCCTC BRAF-1799TA_TPATATTTCTTCATGAAGACCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAG[T/A]GAAATCTCGATGGAGTGGGTCCCATCAGTTTGAACAGTTGTCTGGATCCATTTTG CTNNB1-121AG_TPGTATCCACATCCTCTTCCTCAGGATTGCCTTTACCACTCAGAGAAGGAGCTGTGG[A/G]AGTGGCACCAGAATGGATTNCAGAGTNCAGGTAAGACTGTTGCTGCCAGTGACTA CTNNB1-134CT_TPCAGGACTTGGGAGGTATCCACATCCTCTTCCTCAGGATTGCCTTTACCACTCAGA[C/T]AAGGAGCTGTGGTAGTGGCACCAGAATGGATTNCAGAGTNCAGGTAAGACTGTTG EGFR-2369CT_TPCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCA[C/T]GCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACAATA EGFR-2573TG_TPTTACTTTGCCTCCTTCTGCATGGTATTCTTTCTCTTCCGCACCCAGCAGTTTGGCC[C/T]GCCCAAAATCTGTGATCTTGACATGCTGCGGTGTTTTCACCAGTACGTTCCTGG KRAS-176CG_TPAGGAAGCAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGACACAG[T/G]AGGTCANGAGGAGTACAGTGCAATGAGGGACCAGTACATGAGGACTGGGGAGGGC KRAS-183AC_TPAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGACACAGCAGGTCA[A/C]GAGGAGTACAGTGCAATGAGGGACCAGTACATGAGGACTGGGGAGGGCTTTCTTT KRAS-34GA_TPCCACAAAATGATTCTGAATTAGCTGTATCGTCAAGGCACTCTTGCCTACGCCAC[G/A]AGCTCCAACTACCACAAGTTTATATTCAGTCATTTTCAGCAGGCCTTATAATAAAA KRAS-35GA_TPTCCACAAAATGATTCTGAATTAGCTGTATCGTCAAGGCACTCTTGCCTACGCCA[G/A]CAGCTCCAACTACCACAAGTTTATATTCAGTCATTTTCAGCAGGCCTTATAATAAA KRAS-3GA_TPATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTG[G/A]CGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGA NRAS-181CA_TPAACAAGTGGTTATAGATGGTGAAACCTGTTTGTTGGACATACTGGATACAGCTGGA[C/A]AAGAAGAGTACAGTGCCATGAGAGACCAATACATGAGGACAGGCGAAGGCTTCC NRAS-183AT_TPCAAGTGGTTATAGATGGTGAAACCTGTTTGTTGGACATACTGGATACAGCTGGACA[A/T]GAAGAGTACAGTGCCATGAGAGACCAATACATGAGGACAGGCGAAGGCTTCCTC

The results of cast-PCR using non-tailed ASP and cast-PCR usingtailed-ASP are summarized in FIG. 13. In the cast-PCR reactions havingno tailed primers, the average ΔCt of the 12 tested mutations was 10.3,whereas in the cast-PCR reactions with tailed primers the average ΔCt of12 tested assays is 16.3. Thus, the average ΔΔCt between cast-PCRcomprising tailed ASP versus cast-PCR comprising non-tailed ASP wasabout 6.0, which is about a 64 fold improvement in specificity forreactions comprising ASP +tail primers.

Example 13 Comparison of Cast-PCR and ASB-PCR

Allelic discrimination for assays using cast-PCR methods was compared toassays using other Allele-Specific PCR with a Blocking reagent (ASB-PCR)methods (see, e.g., Morlan et al., 2009).

cast-PCR assays were performed using the general schema and reactionconditions indicated above, using 1 million copies of plasmid DNAcontaining various SNP target sequences (see Table 10). Assays wereperformed using the general experimental design and reaction conditionsindicated above (see section II of Examples). Assay primers and probeswere designed according to the sequences shown in FIG. 19B-D.

ASB-PCR assays were performed using 1 million copies of plasmid DNAcontaining various SNP target sequences (see Table 10), 900 nMnon-tailed allele-specific primers (Tm 58˜62° C.), 3600 nMallele-specific phosphate blocker (Tm 58˜62° C.), 200 nM locus-specificTaqMan probe (Tm 70˜74° C.), and 900 nM locus-specific primers (Tm60˜63° C.).

ASB-PCR assay primers and probes were designed according to thesequences shown in FIG. 20A-C. The ASB-PCR reactions were incubated in a384-well plate at 95° C. for 10 minutes, followed by 50 cycles of at 92°C. for 20 seconds, 60° C. for 45 seconds. All reactions were run in 4replications in an ABI PRISM 7900HT® Sequence Detection System,according to the manufacturer's instructions.

The results for this example are summarized in FIG. 14. In the ASB-PCRassays, the average ΔCt of 12 different mutations was 14.1. In cast-PCRassays, the average ΔCt of the same 12 mutations was 16.3. The ΔΔCtbetween ASB-PCR and cast-PCR was 2.2, which indicates that thespecificity of cast-PCR was approximately 4.6 fold higher than that ofthe ASB-PCR assay.

Example 14 Comparison of MGB and Phosphate Blocker Probes in Cast-PCR

The use of MGB blocker probes was compared to the use of other types ofblocker probes, such as PO₄ blocker probes (e.g., Morlan et al., 2009),in cast-PCR assays.

All assays were performed using the general cast-PCR schema and reactionconditions indicated above (see Section II in Examples), using 1 millioncopies of plasmid DNA containing various SNP target sequences (see Table10), except that reactions contained either 150 nm allele-specific MGBblocker probes or 150 nm allele-specific 3′-phosphate blocker probes.Assay primers and probes were designed according to the sequences shownin FIG. 19B-D (for cast-PCR using MGB blocker probes) or FIGS. 19B-C andFIG. 20C (for cast-PCR using phosphate blocker probes; “PHOS1” to blockallele-1 and “PHOS2” to block allele-2).

The results of assays with phosphate blocker probes or with MGB blockerprobes are summarized in FIG. 15. In cast-PCR assays performed usingphosphate blocker probes the average ΔCt of 12 different mutations was15.1. In comparison, the average ΔCt for the same 12 mutations usingcast-PCR assays performed with MGB blocker probes was slightly higherand gave a ΔCt of 15.8.

Example 15 Improving the Specificity of Cast-PCR Using LNA Modified ASP

LNA-modified cast-PCR assays were performed using the generalexperimental design and reaction conditions indicated above (see SectionII in Examples), using 0.5 ng/μL of genomic DNA. Assay primers andprobes were designed according to the sequences shown in FIGS. 21A-C.For each SNP analyzed, the blocker probes, locus-specific probes andlocus-specific primers were the same and only the allele-specificprimers varied (i.e., with or without an LNA-modification at the 3′end).

The effect of LNA modification of the ASP on the specificity of cast-PCRis summarized in FIG. 16. For the 12 cast-PCR assays performed usingLNA-modified allele-specific primers, the average ΔCt was 16.3. Incomparison, the average ΔCt for the same 12 mutations using cast-PCRassays performed allele-specific primers having no modifications the ΔCtwas noticeably higher at 18.5. Based on the ΔΔCt the assay specificityincreased by approximately 4 fold for those assays that usedLNA-modified allele-specific primers.

Example 16 Improving the Specificity of Cast-PCR Using Other ModifiedASP

cast-PCR assays using other chemically-modified ASPs were performedusing the general experimental design and reaction conditions indicatedabove conditions indicated above (see Section II in Examples), performedin the presence of 1 million copies of plasmid DNA containing variousSNP target sequences (see Table 10). Assay primers and probes weredesigned according to the sequences shown in FIG. 22. For each SNPanalyzed, the blocker probes, locus-specific probes and locus-specificprimers were the same and only the allele-specific primers varied (i.e.,with or without chemical modifications, i.e., ppA, ppG, iso dC or fdU,at the 3′ end).

The results of cast-PCR assays using unmodified ASP and cast-PCR assayswith modified ASP are summarized in FIG. 17. As shown, allele-specificprimers having pyrophosphate modifications (ppA or ppG) at their 3′-endsincreased ΔCt by 2-3, which is approximately a 4-6 fold increase inassay specificity.

It is noted that, as used in this specification and the appended claims,the singular forms “a,” “an,” and “the,” include plural referents unlessexpressly and unequivocally limited to one referent. The use of “or”means “and/or” unless stated otherwise. The use of “comprise,”“comprises,” “comprising,” “include,” “includes,” and “including” areinterchangeable and not intended to be limiting. Furthermore, where thedescription of one or more embodiments uses the term “comprising,” thoseskilled in the art would understand that, in some specific instances,the embodiment or embodiments can be alternatively described using thelanguage “consisting essentially of” and/or “consisting of.”

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises are hereby expressly incorporated by reference intheir entirety for any purpose. In the event that one or more of theincorporated documents defines a term that contradicts that term'sdefinition in this application, this application controls.

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated byreference in their entirety. In the event that one or more of theincorporated literature and similar materials differs.

We claim:
 1. A method for detecting a first allelic variant of a targetsequence in a nucleic acid sample suspected of comprising at least asecond allelic variant of the target sequence, comprising: a) forming afirst reaction mixture by combining: i) the nucleic acid sample; ii) afirst allele-specific primer, wherein an allele-specific nucleotideportion of the first allele-specific primer is complementary to thefirst allelic variant of the target sequence, wherein the firstallele-specific primer does not have a locked nucleic acid (LNA) base;iii) a first allele-specific blocker probe that is complementary to aregion of the target sequence comprising the second allelic variant,wherein said region encompasses a position corresponding to the bindingposition of the allele-specific nucleotide portion of the firstallele-specific primer, and wherein the first allele-specific blockerprobe comprises a minor groove binder at the 3′-end, the 5′-end and/orat an internal position within said allele-specific blocker probe; iv) afirst locus-specific primer that is complementary to a region of thetarget sequence that is 3′ from the first allelic variant and on theopposite strand; and v) a first detector probe; b) carrying out anamplification reaction on the first reaction mixture using the firstlocus-specific primer and the first allele-specific primer to form afirst amplicon; and c) detecting the first amplicon by detecting achange in a detectable property of the first detector probe, therebydetecting the first allelic variant of the target gene in the nucleicacid sample.
 2. The method of claim 1, further comprising using thechange in a detectable property of the first detector probe toquantitate the first allelic variant.
 3. The method of claim 1, furthercomprising: d) forming a second reaction mixture by combining: i) thenucleic acid sample; ii) a second allele-specific primer, wherein anallele-specific nucleotide portion of the second allele-specific primeris complementary to the second allelic variant of the target sequence;iii) a second allele-specific blocker probe that is complementary to aregion of the target sequence comprising the first allelic variant,wherein said region encompasses a position corresponding to the bindingposition of the allele-specific nucleotide portion of the secondallele-specific primer, and wherein the second allele-specific blockerprobe comprises a minor groove binder at the 3′-end, the 5′-end and/orat an internal position within said allele-specific blocker probe; iv) asecond locus-specific primer that is complementary to a region of thetarget sequence that is 3′ from the second allelic variant and on theopposite strand; and v) a second detector probe; e) carrying out anamplification reaction on the second reaction mixture using the secondallele-specific primer and the locus-specific primer, to form a secondamplicon; and f) detecting the second amplicon by detecting a change ina detectable property of the detector probe, thereby detecting thesecond allelic variant of the target gene in the nucleic acid sample. 4.The method of claim 3, further comprising comparing the change in adetectable property of the first detector probe in the first reactionmixture to the change in a detectable property of the second detectorprobe in the second reaction mixture.
 5. The method of claim 3, whereinsaid first, second or first and second allele-specific primer and/orsaid first, second, or first and second allele-specific blocker probecomprises at least one modified base.
 6. The method of claim 5, whereinsaid modified base is an 8-aza-7-deaza-dN (ppN) base analog, where N isadenine (A), cytosine (C), guanine (G), or thymine (T).
 7. The method ofclaim 5, wherein said modified base is a fdU or iso dC base.
 8. Themethod of claim 5, wherein said modified base is any modified base thatincreases the Tm between matched and mismatched target sequences ornucleotides.
 9. The method of claim 5, wherein said modified base islocated at (a) the 3′-end, (b) the 5′-end, (c) at an internal positionor at any combination of (a), (b) or (c) within said allele-specificprimer and/or allele-specific blocker probe.
 10. The method of claim 5,wherein the specificity of said detecting is improved at least 2 fold bythe inclusion of said modified base in said first, second or first andsecond allele-specific primer and/or said first, second, or first andsecond allele-specific blocker probe as compared to when it is not. 11.The method of claim 3, wherein said carrying out an amplificationreaction comprises a 2-stage cycling protocol.
 12. The method of claim11, wherein the number of cycles in the first stage of said 2-stagecycling protocol comprises fewer cycles than the number of cycles usedin the second stage.
 13. The method of claim 11, wherein said number ofcycles in the first stage is about 90% fewer cycles than said number ofcycles in the second stage.
 14. The method of claim 11, wherein saidnumber of cycles in the first stage is between 3-7 cycles and saidnumber of cycles in the second stage is between 42-48 cycles.
 15. Themethod of claim 11, wherein the annealing/extension temperature usedduring the first cycling stage of said 2-stage cycling protocol isbetween 1-3° C. lower than the annealing/extension temperature usedduring the second stage.
 16. The method of claim 11, wherein saidannealing/extension temperature used during the first cycling stage ofsaid 2-stage cycling protocol is between 56-59° C. and saidannealing/extension temperature used during said second stage is between60-62° C.
 17. The method of claim 1, wherein said step (a) is precededby a pre-amplification step.
 18. The method of claim 17, wherein saidpre-amplification step comprises a multiplex amplification reaction thatuses at least two complete sets of allele-specific primers andlocus-specific primers, wherein each set is suitable or operative foramplifying a specific polynucleotide of interest.
 19. The method ofclaim 18, wherein the products of said multiplex amplification reactionare divided into secondary single-plex amplification reactions, whereineach single-plex amplification reaction contains at least one primer setpreviously used in said multiplex reaction.
 20. The method of claim 18,wherein said multiplex amplification reaction further comprises aplurality of allele-specific blocker probes.